Hydrolytically stable maleimide-terminated polymers

ABSTRACT

The present invention is directed to hydrolytically stabilized maleimide-functionalized water soluble polymers and to methods for making and utilizing such polymers and their precursors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S.provisional application Serial No. 60/437,211, filed Dec. 31, 2002,which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to particular maleimide-terminated watersoluble polymers, and to methods for making and utilizing such polymers.In particular, the invention relates to: (i) hydrolytically stabilizedpolymers having one or more terminal maleimide groups, (ii) conjugatesformed from the attachment of a maleimide-terminated water solublepolymer reagent as described herein to another substance, such as anactive agent or surface, (iii) methods for synthesizing such polymericreagents, (iv) compositions comprising the polymeric reagents, and thelike.

BACKGROUND OF THE INVENTION

[0003] Due to recent advances in biotechnology, therapeutic proteins andother biomolecules, e.g. antibodies and antibody fragments, can now beprepared on a large scale, making such biomolecules more widelyavailable. Unfortunately, the clinical usefulness of potentialtherapeutic biomolecules is often hampered by their rapid proteolyticdegradation, low bioavailability, instability upon manufacture, storageor administration, or by their immunogenicity. Due to the continuedinterest in administering proteins and other biomolecules fortherapeutic use, various approaches to overcoming these deficiencieshave been explored.

[0004] One such approach which has been widely explored is themodification of proteins and other potentially therapeutic molecules bycovalent attachment of a water soluble polymer such as polyethyleneglycol or “PEG” (Abuchowski, A., et al, J. Biol. Chem. 252 (11), 3579(1977); Davis, S., et al., Clin. Exp Immunol., 46, 649-652 (1981). Thebiological properties of PEG-modified proteins, also referred to asPEG-conjugates or pegylated proteins, have been shown, in many cases, tobe considerably improved over those of their non-pegylated counterparts(Herman, et al., Macromol. Chem. Phys., 195, 203-209 (1994).Polyethylene glycol-modified proteins have been shown to possess longercirculatory times in the body due to increased resistance to proteolyticdegradation, and also to possess increased thermostability (Abuchowski,A., et al., J. Biol. Chem., 252, 3582-3586 (1977). A similar increase inbioefficacy is observed with other biomolecules, e.g. antibodies andantibody fragments (Chapman, A., Adv. Drug Del. Rev. 54, 531-545(2002)).

[0005] Typically, attachment of polyethylene glycol to a drug or othersurface is accomplished using an activated PEG derivative, that is tosay, a PEG having at least one activated terminus suitable for reactionwith a nucleophilic center of a biomolecule (e.g., lysine, cysteine andsimilar residues of proteins). Most commonly employed are methods basedupon the reaction of an activated PEG with protein amino groups, such asthose present in the lysine side chains of proteins. Polyethylene glycolhaving activated end groups suitable for reaction with the amino groupsof proteins include PEG-aldehydes (Harris, J. M., Herati, R. S., PolymPrepr. (Am. Chem. Soc., Div. Polym. Chem), 32(1), 154-155 (1991), mixedanhydrides, N-hydroxysuccinimide esters, carbonylimadazolides, andchlorocyanurates (Herman, S., et al., Macromol. Chem. Phys. 195, 203-209(1994)). Although many proteins have been shown to retain activityduring PEG modification, in some instances, polymer attachment throughprotein amino groups can be undesirable, such as when derivatization ofspecific lysine residues inactivates the protein (Suzuki, T., et al.,Biochimica et Biophysica Acta 788, 248-255 (1984)). Moreover, since mostproteins possess several available/accessible amino groups, the polymerconjugates formed are typically mixtures of mono-pegylated,di-pegylated, tri-pegylated species and so on, which can be difficultand also time-consuming to characterize and separate. Further, suchmixtures are often not reproducibly prepared, which can create problemsduring scale-up for regulatory approval and subsequentcommercialization.

[0006] One method for avoiding these problems is to employ asite-selective polymer reagent that targets functional groups other thanamines. One particularly attractive target is the thiol group onproteins, present in the amino acid, cysteine. Cysteines are typicallyless abundant in proteins than lysines, thus reducing the likelihood ofprotein deactivation upon conjugation to these thiol-containing aminoacids. Moreoever, conjugation to cysteine sites can often be carried outin a well-defined manner, leading to the formation of single speciespolymer-conjugates.

[0007] Polyethylene glycol derivatives having a thiol-selective reactiveend group include maleimides, vinyl sulfones, iodoacetamides, thiols,and disulfides, with maleimides being the most popular. Thesederivatives have all been used for coupling to the cysteine side chainsof proteins (Zalipsky, S. Bioconjug. Chem. 6, 150-165 (1995); Greenwald,R. B. et al. Crit. Rev. Ther. Drug Carrier Syst. 17, 101-161 (2000);Herman, S., et al., Macromol. Chem. Phys. 195, 203-209 (1994)). However,many of these reagents have not been widely exploited due to thedifficulty in their synthesis and purification.

[0008] As discussed above, polyethylene glycol derivatives having aterminal maleimide group are one of the most popular types ofsulfhydryl-selective reagents, and are commercially available from anumber of sources. Although not widely appreciated or recognized, theApplicants have recognized that many PEG-maleimides unfortunately arehydrolytically unstable during storage and conjugation to a drugcandidate. More particularly, a substantial degree of hydrolysis of themaleimide ring has been observed, both prior to and after conjugation.This instability can result in the formation of multiple species of drugconjugates within a drug-conjugate composition. The various drugconjugate species are likely to possess similar biological activities,but may differ in their pharmacokinetic properties, making suchcompositions undesirable for patient administration. Additionally,separation of the open-ring and closed-ring forms of the drug conjugatecan be extremely difficult to carry out. Moreover, such hydrolyticinstability can lead to inconsistency in drug batches. Thus, theapplicants have realized a continuing need in the art for thedevelopment of new activated PEGs useful for coupling to biologicallyactive molecules, desirably in a site-selective fashion, that are stableduring both storage and coupling. This invention meets those needs.

SUMMARY OF THE INVENTION

[0009] The present invention provides a unique family of hydrolyticallystabilized maleimide-terminated polymers, where the polymers compriseparticular linkers interposed between a polymer segment and a maleimidegroup.

[0010] The invention is based on the discovery that the incorporation ofa saturated acyclic, cyclic, or alicyclic hydrocarbon linker adjacent tothe maleimide ring of a maleimide-terminated polymer substantiallyreduces its instability. Provided herein are polymers having ahydrolytically stabilized maleimide ring, their polymer precursors,conjugates of the hydrolytically stabilized maleimide-terminatedpolymers, and methods for making and using such polymers and theirconjugates.

[0011] Generally, the present invention is directed to a water solublepolymer having the structure:

[0012] In the generalized structure above, POLY is a water solublepolymer segment, and L is a linkage that imparts hydrolytical stabilityto the adjacent maleimide ring. Typically the linker comprises asaturated acyclic, cyclic or alicyclic hydrocarbon chain adjacent to themaleimide ring and contains a total of about 3 to about 20 carbon atoms,optionally containing other non-interfering atoms or functional groups.

[0013] More particularly, in one aspect, the invention is directed to awater-soluble polymer having the structure:

[0014] In structure II, POLY is a water-soluble polymer segment, b is 0or 1, and X is a hydrolytically stable linker comprising at least 3contiguous saturated carbon atoms. Preferably, the polymer is absentaromatic groups and ester linkages. In one embodiment, POLY is directlycovalently bonded to the amide carbonyl carbon, optionally via anintervening oxygen ([O]_(b)) to form a carbamate group. In analternative embodiment, POLY is connected to the amide carbonyl carbon,optionally via an intervening oxygen, ([O]) in the instance when b=1,via an intervening spacer, for example, a methylene.

[0015] In one embodiment, X is a saturated acyclic, cyclic or alicyclichydrocarbon chain having a total of about 3 to about 20 carbon atoms.More particularly, X can possess a total number of carbon atoms selectedfrom the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, and 20. Preferred ranges for total number of carbonatoms in the linker X are from about 3 to about 20, or from about 4 toabout 12, or from about 4 to about 10, or from about 5 to about 8 atoms.

[0016] The linker X in formula II may possess any of a number ofstructural features. In one embodiment, X is a linear saturated acyclichydrocarbon chain. In yet another embodiment, X is a branched saturatedacyclic hydrocarbon chain and can contain one or even two substituents,at any one or more of the carbon positions in the chain. For example, Xcan be branched at the carbon α to the maleimidyl group, or at thecarbon β to the maleimidyl group, or at the carbon γ to the maleimidylgroup. For hydrocarbon chains having up to 19 carbon atoms, any one ofpositions 1 to 19 (with position 1 being the one proximal to themaleimide ring) may be branched. For instance, for an exemplarysaturated hydrocarbon chain having from 2 to 19 carbon atoms designatedC₁-C₂-C₃-C₄-C₅-C₆-C₇-C₈-C₉-C₁₀-C₁₁-C₁₂-C₁₃-C₁₄-C₁₅-C₁₆-C₁₇-C₁₈-C₁₉-, anyone or more of carbons C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, or C₁₉, depending on the total numberof carbons in the chain, may be branched. Preferably, in any givensaturated hydrocarbon chain or alicyclic linker, 4 or fewer carbon atomsare branched, with the overall number of branching positions preferablyequal to 1, 2, 3, or 4. Embodiments wherein the “branch” points takentogether form a saturated ring or ring system (e.g., bicyclic,tricyclic, etc.) are discussed separately below.

[0017] Representative polymers in accordance with different embodimentsof the invention are provided below.

[0018] For example, in structure III, y is an integer from 1 to about20; and R¹ and R² in each occurrence are each independently H or anorganic radical that is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl.

[0019] Preferably, in structure III above, R¹ and R² in each occurrenceare each independently H or an organic radical selected from the groupconsisting of lower alkyl and lower cycloalkyl. Y is preferably selectedfrom the group consisting of 3, 4, 5, 6, 7, 8, 9, and 10. In aparticular embodiment of structure III, R¹ and R² are both H.

[0020] Various embodiments of structure III include the following.

[0021] In illustrative structure III-A, at least one of R¹ or R² onC_(α) is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl, and y is at least one and may possess any of theabove-described specific values.

[0022] Particular embodiments of structure I-A include those where:

[0023] (i) each of R¹ and R on C_(α) is independently selected from thegroup consisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl,and/or

[0024] (ii) all other non-C_(α) R¹ and R² variables are H, and/or

[0025] (iii) at least one of R¹ or R² on C_(α) is lower alkyl or lowercycloalkyl, and/or

[0026] (iv) R² on C_(α) is H, and/or

[0027] (v) R¹ on C_(α) is selected from the group consisting of methyl,ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl,and methylenecyclohexyl.

[0028] Yet another particular embodiment of this aspect of the inventionis provided as structure III-B below.

[0029] wherein R¹ and R² are each independently alkyl or cycloalkyl.Alternatively, R¹ is alkyl or cycloalkyl and R is H. Additionalembodiments of structure III-B are those where (i) R¹ and R² are eachindependently either methyl or ethyl, and/or R¹ and R² are the same.

[0030] In yet another embodiment of structure m, a polymer of theinvention possesses the following structure:

[0031] where R¹ and R² are each independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, butare not both H, and y is at least 2.

[0032] Particular embodiments of this structure include those where (i)R¹ and R² are each independently H, lower alkyl or lower cycloalkyl,and/or (ii) R¹ and R² are each independently selected from the groupconsisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,pentyl, cyclopentyl, hexyl, and cyclohexyl, and/or R² is H.

[0033] In yet another embodiment of structure III,

[0034] at least one of R¹ and R² attached to C_(γ) is selected from thegroup consisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl.Specific embodiments include those where: (i) at least one of R¹ and R²attached to C_(γ) is alkyl or cycloalkyl and all other R¹ and R²variables are H, and/or (ii) one of the R¹ variables attached to C_(α)or C_(β) is alkyl or cycloalkyl, and all other R¹ and R² variables areH.

[0035] As described previously, X can be a saturated cyclic or alicyclichydrocarbon chain, that is to say, the linker X may contain one or morecyclic hydrocarbons. Generally, also provided herein are polymers havingthe structure:

[0036] In the preceding structure, CYC_(a) is a cycloalkylene grouphaving “a” ring carbons, where the value of “a” ranges from 3 to 12; andp and q are each independently 0 to 20, and p+q+a≦20. R¹ and R², in eachoccurrence, are each independently H or an organic radical that isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.

[0037] CYC_(a) in accordance with the invention encompasses unicyclic,bicyclic, tricyclic structures and the like.

[0038] Various embodiments of structure IV above include those where:

[0039] (i) p and q are each independently selected from the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8, and/or

[0040] (ii) R¹, in each occurrence, is independently H or an organicradical that is either lower alkyl or lower cycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is eitherlower alkyl or lower cycloalkyl, and/or

[0041] (iii) a is selected from the group consisting of 5, 6, 7, 8 and9, and/or

[0042] (iv) a is 6 and CYC_(a) is a 1,1-, 1,2-, 1,3- or 1,4-substitutedcyclohexyl ring, and/or

[0043] (v) p and q each independently range from 0 to 4, and/or

[0044] (vi) R¹ and R² are H in every occurrence.

[0045] For linkers comprising a cyclcoalkylene group and twosubstituents thereon, the substituents can be either cis or trans.

[0046] Specific embodiments of structure IV include:

[0047] wherein q and p are as described above. In a particularembodiment, q and p each independently range from 0 to 6. In yet anotherembodiment, q ranges from 0 to 6 and p is zero.

[0048] Yet another exemplary polymer structure having a cycloalkylenering in accordance with the invention is:

[0049] wherein q and p are as defined above, and more preferably, eachindependently range from 0 to 6.

[0050] Polymers of the invention include monofunctional, bifunctional,and multi-functional structures.

[0051] For instance, a polymer of the invention may be describedgenerally by the following structure:

[0052] where X and b are as previously defined, b′ is 0 or 1, and X′ isa hydrolytically stable linker comprising at least 3 contiguoussaturated carbon atoms. In the above embodiment, b and b′ may be thesame of different, and X and X′ may be the same or different. In oneparticular embodiment the polymer reagent is homo-bifunctional, that isto say, both reactive end groups are the same. In this instance, bequals b′ and X equals X′.

[0053] Preferably, the water-soluble polymer segment in any of thepolymer maleimides provided herein is a poly(alkylene oxide), apoly(vinyl pyrrolidone), a poly(vinyl alcohol), a polyoxazoline, apoly(acryloylmorpholine), or a poly(oxyethylated polyol). In a preferredembodiment, the polymer segment is a poly(alkylene oxide), preferablypoly(ethylene glycol).

[0054] According to one embodiment, the poly(ethylene glycol) segmentcomprises the structure: Z-(CH₂CH₂O)_(n)—CH₂CH₂—, where n ranges fromabout 10 to about 4000 and Z is a moiety comprising a functional groupselected from the group consisting of hydroxy, amino, ester, carbonate,aldehyde, alkenyl, acrylate, methacrylate, acrylamide, sulfone, thiol,carboxylic acid, isocyanate, isothiocyanate, hydrazide, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, alkoxy,benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein. In thisembodiment, Z comprises a reactive functional group or an end-cappinggroup.

[0055] In yet a more specific embodiment, POLY may be terminally cappedwith an end-capping moiety such as alkoxy, substituted alkoxy,alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy,aryloxy, substituted aryloxy, or phospholipid. Preferred end-cappinggroups include methoxy, ethoxy, and benzyloxy.

[0056] Generally, POLY possesses a nominal average molecular massfalling within one of the following ranges: from about 100 daltons toabout 100,000 daltons, from about 1,000 daltons to about 50,000 daltons,or from about 2,000 daltons to about 30,000 daltons. Preferred molecularmasses for POLY include 250 daltons, 500 daltons, 750 daltons, 1 kDa, 2kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 40 kDa, and 50 kDa, or evengreater.

[0057] The polymer segment may possess any of a number of geometries,e.g., may be linear, branched or forked.

[0058] The polymer of the invention may be multi-armed. An exemplarymulti-arm polymer in accordance with the invention has the structure:

[0059] In the above illustrative structure, d is an integer from 3 toabout 100, and R is a residue of a central core molecule having 3 ormore hydroxyl groups, amino groups, or combinations thereof. Preferably,d is an integer from 3 to about 12.

[0060] In an alternative multi-arm embodiment, the polymer correspondsto the structure:

[0061] In this structure,

[0062] PEG is —(CH₂CH₂O)_(n)CH₂CH₂—,

[0063] M is:

[0064] and m is selected from the group consisting of 3, 4, 5, 6, 7, and8.

[0065] In yet another aspect, provided herein are polymers having theabove described features and exemplary structures, with the exceptionthat the maleimide group in the above-described polymers is replacedwith an amino group, preferably a primary amino, —NH₂. Such polymers areuseful not only as activated polymer reagents, e.g., for conjugation toan active agent, but are also precursors to the stabilized maleimidepolymers of the invention.

[0066] For example, the invention encompasses a water-soluble polymer inaccordance with the structure:

[0067] where the variables X and b are as previously described, bothgenerally and in specific embodiments. Preferably, the polymer is absentaromatic groups and ester linkages.

[0068] In yet another aspect, the invention provides a water solublepolymer having the structure:

[0069] where POLY is a water-soluble polymer segment, and X is ahydrolytically stable linker that is a saturated cyclic or alicyclichydrocarbon chain having a total of about 3 to about 20 carbon atoms.Preferably, the polymer is absent aromatic groups and ester linkages.

[0070] Polymers in accordance with this aspect of the inventionencompass, in various embodiments, those where X corresponds to thegeneral and specific cyclic and alicyclic hydrocarbon structuresdescribed herein.

[0071] In one particular embodiment of structure XIII, the linker X hasthe structure:

[0072] where

[0073] CYC_(a) is a cycloalkylene group having “a” ring carbons, wherethe value of “a” ranges from 3 to 12; p and q are each independently 0to 20, and p+q+a≦20. In structure XIII-A, each of R¹ and R², in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl. In instances where CYC_(a) possesses only twosubstituents, such substituents can be cis or trans.

[0074] In yet another embodiment of structure XIII-A, p and q are eachindependently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,7, and 8.

[0075] In yet another embodiment of structure XIII-A, R¹, in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of lower alkyl, lower cycloalkyl, and loweralkylenecycloalkyl, and R², in each occurrence, is independently H or anorganic radical that is selected from the group consisting of loweralkyl, lower cycloalkyl, and lower alkylenecycloalkyl.

[0076] In yet another embodiment of structure XIII-A, a is selected fromthe group consisting of 5, 6, 7, 8 and 9.

[0077] In a preferred embodiment of structure XIII-A, a is 6 and CYC_(a)is a 1,1-, 1,2-, 1,3- or 1,4-substituted cyclohexyl ring. Additionalembodiments include those where p and q each independently range from 0to 4, and/or where R¹ and R² are H in every occurrence.

[0078] Particularly, certain embodiments of structure XIII include:

[0079] 1

[0080] wherein q and p each independently range from 0 to 6.

[0081] In yet another embodiment of structure XIII, CYC_(a) is bicyclicor tricyclic.

[0082] In yet another aspect, the invention includes hydrogels preparedusing any one or more of the polymers described herein.

[0083] In yet another aspect, provided is a method for forming ahydrolytically stable maleimide-terminated polymer. The method includesthe steps of (a) reacting a polymer having the structure,POLY-[O]_(b)—C(O)-LG (IX), with a diamine having the structure,NH₂—X—NH₂ (XII), under conditions effective to formPOLY-[O]_(b)—C(O)—HN—X—NH₂ (X), followed by (b) convertingPOLY-[O]_(b)—C(O)—HN—X—NH₂ (X) into POLY-[O]_(b)—C(O)—HN—X-MAL (II).

[0084] The variables POLY, b and X are as described previously, bothgenerally and specifically, LG represents a leaving group, and MAL ismaleimide. Preferably, the resulting product,POLY-[O]_(b)—C(O)—HN—X-MAL, is absent aromatic groups and esterlinkages.

[0085] The method can be used to prepare any of the polymer-terminatedmaleimides described herein.

[0086] Preferred leaving groups include halide, N-hydroxysuccinimide,N-hydroxybenzotriazole, para-nitrophenolate.

[0087] In one embodiment of the method, one of the amino groups in saidNH₂—X—NH₂ reagent is in protected form. In this instance, the methodwill generally comprise, after the reacting step, deprotecting the aminogroup in POLY-[O]_(b)—C(O)—H₂N—X—NH₂.

[0088] The reacting step is typically carried out in an organic solvent.Typical solvents include acetonitrile, chlorinated hydrocarbons,aromatic hydrocarbons, tetrahydrofuran (THF), dimethylformamide (DMF),and dimethylsulfoxide.

[0089] In another embodiment of the method, the reacting step isconducted under an inert atmosphere such as nitrogen or argon.

[0090] Temperatures for carrying out the reacting step range from about0 to 100° C.

[0091] In a further embodiment, the reacting step is carried out in thepresence of a base. Examplary bases include triethyl amine and othersimilar tertiary amines, pyridine, 4-(dimethylamino)pyridine, andinorganic bases such as sodium carbonate.

[0092] In a preferred embodiment, the method further includes the stepof purifying the product from step (a) prior to the converting step, forexample, by column chromatography, preferably by ion exchangechromatography.

[0093] In yet another specific embodiment, the converting step comprisesreacting POLY-[O]_(b)—C(O)—H₂N—X—NH₂ with a reagent selected from thegroup consisting of N-methoxycarbonylmaleimide,exo-7-oxa[2.2.1]bicycloheptane-2,3-dicarboxylic anhydride, and maleicanhydride, under conditions suitable for formingPOLY-[O]_(b)—C(O)—H₂N—X-MAL in a reaction mixture.

[0094] In an embodiment where the reagent is N-methoxycarbonylmaleimide,the converting step is preferably carried out in water or an aqueousmixture of water and a water miscible solvent such as acetone oracetonitrile.

[0095] In an embodiment of the above method where the reagent is maleicanhydride, the converting step comprises reactingPOLY-[O]_(b)—C(O)—H₂N—X—NH₂ with maleic anhydride under conditionseffective to form POLY-[O]_(b)—C(O)—NH—X—NH—C(O)CH═CHCOOH (XI) as anintermediate, followed by heatingPOLY-[O]_(b)—C(O)—H₂N—X—NH—C(O)CH═CHCOOH under conditions effective topromote cyclization by elimination of water to formPOLY-[O]_(b)—C(O)—NH—X-MAL.

[0096] Generally, the method further comprises the step of recoveringthe product, POLY-[O]_(b)—C(O)—H₂N—X-MAL, from the reaction mixture.

[0097] Preferably, the recovered product has a purity of greater thanabout 80%, and is absent polymeric impurities other than the desiredproduct.

[0098] Exemplary diamines for carrying out the method include

[0099] where the variables encompass those both generally andspecifically described above.

[0100] In yet another aspect, provided herein is an alternative methodfor preparing a hydrolytically stable maleimide-terminated polymer ofthe invention. The method includes the steps of reactingPOLY-[O]_(b)—C(O)-LG (IX) with H₂N—X-MAL (XIV) under conditionseffective to form POLY-[O]_(b)—C(O)—HN—X-MAL (II), where the variablesPOLY, b, X, LG, and MAL are as previously defined, both generally andspecifically, regardless of the subject embodiment used forexemplification purposes.

[0101] In yet another aspect, the invention provides a conjugate formedby reaction of a biologically active agent with any of the hereindescribed hydrolytically stable maleimide- or amino-terminated polymers.

[0102] More particularly, one embodiment of this aspect of the inventionincludes a conjugate comprising the following structure:

[0103] where the variables POLY, b and X are as previously defined, bothgenerally and specifically, regardless of the subject exemplifyingembodiment, “POLY-[O]_(b)—C(O)—NH—X—” is absent aromatic groups andester linkages, and “—S-biologically active agent” represents abiologically active agent comprising a thiol (—SH) group.

[0104] In one embodiment, provided is a composition comprising the aboveconjugate. In a more particular embodiment, the conjugate compositioncomprises a single polymer conjugate species.

[0105] In yet another embodiment, the invention is directed to aconjugate comprising the following structure:

[0106] wherein POLY, b, and X are as defined above, both generally andspecifically, “POLY-[O]_(b)—C(O)—NH—X—” is absent aromatic groups andester linkages, and “—NH— biologically active agent” represents abiologically active agent comprising an amino group.

[0107] In yet another related aspect, the invention provides a methodfor forming a polymer conjugate, where the method includes the step ofcontacting a biologically active agent comprising a reactive thiolgroup, “HS-biologically active agent”, with a hydrolytically ring stablemaleimide terminated polymer of the invention, under conditionseffective to promote formation of a polymer conjugate having thestructure:

[0108] These and other objects and features of the invention will becomemore fully apparent when read in conjunction with the following figuresand detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0109]FIGS. 1A and 1B provide structures of exemplary polymer maleimidesof the invention containing hydrolytically stable cyclic (includingbicyclic and tricyclic) linkers, and

[0110]FIG. 2 provides structures of exemplary diamines useful inpreparing certain stabilized polymer maleimides of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0111] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like as such mayvary. It is also to be understood that the terminology used herein isfor describing particular embodiments only, and is not intended to belimiting.

[0112] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

[0113] Definitions

[0114] The following terms as used herein have the meanings indicated.

[0115] As used in the specification, and in the appended claims, thesingular forms “a”, “an”, “the”, include plural referents unless thecontext clearly dictates otherwise.

[0116] “PEG” or “poly(ethylene glycol)” as used herein, is meant toencompass any water-soluble poly(ethylene oxide). Typically, PEGs foruse in the present invention will comprise one of the two followingstructures: “—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” dependingupon whether or not the terminal oxygen(s) has been displaced, e.g.,during a synthetic transformation. The variable (n) ranges from 3 to3000, and the terminal groups and architecture of the overall PEG mayvary. When PEG further comprises a linker moiety (to be described ingreater detail below), the atoms comprising the linker, when covalentlyattached to a PEG segment, do not result in formation of (i) anoxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N). “PEG” means a polymer that contains amajority, that is to say, greater than 50%, of subunits that are—CH₂CH₂O—. PEGs for use in the invention include PEGs having a varietyof molecular weights, structures or geometries (e.g., branched, linear,forked PEGs, dendritic, and the like), to be described in greater detailbelow.

[0117] “PEG diol”, also known as alpha-,omega-dihydroxylpoly(ethyleneglycol), can be represented in brief form as HO-PEG-OH, where PEG is asdefined above.

[0118] “Water-soluble”, in the context of a polymer of the invention ora “water-soluble polymer segment” is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

[0119] An “end-capping” or “end-capped” group is an inert ornon-reactive group present on a terminus of a polymer such as PEG. Anend-capping group is one that does not readily undergo chemicaltransformation under typical synthetic reaction conditions. An endcapping group is generally an alkoxy group, —OR, where R is an organicradical comprised of 1-20 carbons and is preferably lower alkyl (e.g.,methyl, ethyl) or benzyl. “R” may be saturated or unsaturated, andincludes aryl, heteroaryl, cyclo, heterocyclo, and substituted forms ofany of the foregoing. For instance, an end capped PEG will typicallycomprise the structure “RO—(CH₂CH₂O)_(n)—”, where R is as defined above.Alternatively, the end-capping group can also advantageously comprise adetectable label. When the polymer has an end-capping group comprising adetectable label, the amount or location of the polymer and/or themoiety (e.g., active agent) to which the polymer is coupled, can bedetermined by using a suitable detector. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, colorimetric (e.g., dyes), metal ions, radioactive moieties,and the like. The end-capping group can also advantageously comprise aphospholipid. When the polymer has an end-capping group such as aphospholipid, unique properties (such as the ability to form organizedstructures with similarly end-capped polymers) are imparted to thepolymer. Exemplary phospholipids include, without limitation, thoseselected from the class of phospholipids called phosphatidylcholines.Specific phospholipids include, without limitation, those selected fromthe group consisting of dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine,disteroylphosphatidylcholine, behenoylphosphatidylcholine,arachidoylphosphatidylcholine, and lecithin.

[0120] “Non-naturally occurring” with respect to a polymer of theinvention means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may however contain oneor more subunits or segments of subunits that are naturally occurring,so long as the overall polymer structure is not found in nature.

[0121] “Molecular mass” in the context of a water-soluble polymer of theinvention such as PEG, refers to the nominal average molecular mass of apolymer, typically determined by size exclusion chromatography, lightscattering techniques, or intrinsic velocity determination in1,2,4-trichlorobenzene. The polymers of the invention are typicallypolydisperse, possessing low polydispersity values of less than about1.20.

[0122] The term “reactive” or “activated” refers to a functional groupthat reacts readily or at a practical rate under conventional conditionsof organic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

[0123] “Not readily reactive” or “inert” with reference to a functionalgroup present on a molecule in a reaction mixture, indicates that thegroup remains largely intact under conditions effective to produce adesired reaction in the reaction mixture.

[0124] A “protecting group” is a moiety that prevents or blocks reactionof a particular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

[0125] A functional group in “protected form” refers to a functionalgroup bearing a protecting group. As used herein, the term “functionalgroup” or any synonym thereof is meant to encompass protected formsthereof.

[0126] The term “linker” is used herein to refer to an atom or acollection of atoms optionally used to link interconnecting moieties,such as a polymer segment and a maleimide. The linkers of the inventionare generally hydrolytically stable.

[0127] A “physiologically cleavable” or “hydrolyzable” or “degradable”bond is a relatively weak bond that reacts with water (i.e., ishydrolyzed) under physiological conditions. The tendency of a bond tohydrolyze in water will depend not only on the general type of linkageconnecting two central atoms but also on the substituents attached tothese central atoms. Appropriate hydrolytically unstable or weaklinkages include but are not limited to carboxylate ester, phosphateester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines,orthoesters, peptides and oligonucleotides, thioesters, thiolesters, andcarbonates.

[0128] An “enzymatically degradable linkage” means a linkage that issubject to degradation by one or more enzymes.

[0129] A “hydrolytically stable” linkage or linker, for the purposes ofthe present invention, and in particular in reference to the polymers ofthe invention, refers to an atom or to a collection of atoms, that ishydrolytically stable under normal physiological conditions. That is tosay, a hydrolytically stable linkage does not undergo hydrolysis underphysiological conditions to any appreciable extent over an extendedperiod of time. Examples of hydrolytically stable linkages include butare not limited to the following: carbon-carbon bonds (e.g., inaliphatic chains), ethers, amides, urethanes, amines, and the like.Hydrolysis rates of representative chemical bonds can be found in moststandard chemistry textbooks.

[0130] A “hydrolytically stabilized maleimide ring”, in reference to apolymer of the invention, is one that resists hydrolysis of themaleimide ring in comparison to the ring opening-stability of itslinkerless polymer maleimide counterpart. For example, if a subjectwater soluble maleimide has a structureCH₃O—(CH₂CH₂O)_(5K)—CH₂CH₂—C(O)—NH—CH₂-1,3-C₆H₁₀—CH₂-MAL, where thelinker is —C(O)—NH—CH₂-1,3-C₆H₁₀—CH₂—, then the corresponding linkerlessversion to form a basis for comparison isCH₃O—(CH₂CH₂O)_(5K)—CH₂CH₂-MAL. Typically, such hydrolysis evaluationsare carried out at pH 7.5 in phosphate buffer at room temperature andare measured by observing the UV absorption of the maleimide ring. So, ahydrolytically stabilized maleimide ring contained in a polymer reagentof the invention is one that has a degree of hydrolytic stability thatis improved over that of its linkerless counterpart. Preferably, ahydrolytically stabilized maleimide ring in accordance with theinvention results in a stabilized polymer maleimide having a hydrolysishalf-life under the above-described conditions of at least about 16hours, and more preferably of at least about 20 hours.

[0131] “Branched” in reference to the geometry or overall structure of apolymer refers to polymer having 2 or more polymer “arms”. A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, that for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

[0132] “Branch point” refers to a bifurcation point comprising one ormore atoms at which a polymer splits or branches from a linear structureinto one or more additional polymer arms.

[0133] A “dendrimer” is a globular, size monodisperse polymer in whichall bonds emerge radially from a central focal point or core with aregular branching pattern and with repeat units that each contribute abranch point. Dendrimers exhibit certain dendritic state properties suchas core encapsulation, making them unique from other types of polymers.

[0134] “Substantially” or “essentially” means nearly totally orcompletely, for instance, 95% or greater of some given quantity.

[0135] An “alkyl” or “alkylene” group, depending upon its position in amolecule and the number of points of attachment of the group to atomsother than hydrogen, refers to a hydrocarbon chain or moiety, typicallyranging from about 1 to 20 atoms in length. Such hydrocarbon chains arepreferably but not necessarily saturated unless so indicated and may bebranched or straight chain, although typically straight chain ispreferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl,pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, hexyl, heptyl, andthe like.

[0136] “Lower alkyl” or “lower alkylene” refers to an alkyl or alkylenegroup as defined above containing from 1 to 6 carbon atoms, and may bestraight chain or branched, as exemplified by methyl, ethyl, n-butyl,i-butyl, t-butyl.

[0137] “Cycloalkyl” or “cycloalkylene”, depending upon its position in amolecule and the number of points of attachment to atoms other thanhydrogen, refers to a saturated or unsaturated cyclic hydrocarbon chain,including polycyclics such as bridged, fused, or spiro cyclic compounds,preferably made up of 3 to about 12 carbon atoms, more preferably 3 toabout 8.

[0138] “Lower cycloalkyl” or “lower cycloalkylene” refers to acycloalkyl group containing from 1 to 6 carbon atoms.

[0139] “Alicyclic” refers to any aliphatic compound that contains a ringof carbon atoms. An alicyclic group is one that contains a “cycloalkyl”or “cycloalkylene” group as defined above that is substituted with oneor more alkyl or alkylenes.

[0140] “Non-interfering substituents” are those groups that, whenpresent in a molecule, are typically non-reactive with other functionalgroups contained within the molecule.

[0141] The term “substituted” as in, for example, “substituted alkyl,”refers to a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

[0142] “Alkoxy” refers to an —O—R group, wherein R is alkyl orsubstituted alkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy,propyloxy, benzyl, etc.), preferably C₁-C₇.

[0143] As used herein, “alkenyl” refers to a branched or unbranchedhydrocarbon group of 1 to 15 atoms in length, containing at least onedouble bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl,isobutenyl, octenyl, decenyl, tetradecenyl, and the like.

[0144] The term “alkynyl” as used herein refers to a branched orunbranched hydrocarbon group of 2 to 15 atoms in length, containing atleast one triple bond, ethynyl, n-propynyl, isopropynyl, n-butynyl,isobutynyl, octynyl, decynyl, and so forth.

[0145] “Aryl” means one or more aromatic rings, each of 5 or 6 corecarbon atoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

[0146] “Heteroaryl” is an aryl group containing from one to fourheteroatoms, preferably N, O, or S, or a combination thereof. Heteroarylrings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

[0147] “Heterocycle” or “heterocyclic” means one or more rings of 5-12atoms, preferably 5-7 atoms, with or without unsaturation or aromaticcharacter and having at least one ring atom which is not a carbon.Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0148] “Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

[0149] “Substituted heterocycle” is a heterocycle having one or moreside chains formed from non-interfering substituents.

[0150] “Electrophile” refers to an ion, atom, or collection of atomsthat may be ionic, having an electrophilic center, i.e., a center thatis electron seeking, capable of reacting with a nucleophile.

[0151] “Nucleophile” refers to an ion or atom or collection of atomsthat may be ionic, having a nucleophilic center, i.e., a center that isseeking an electrophilic center, and capable of reacting with anelectrophile.

[0152] “Active agent” as used herein includes any agent, drug, compound,composition of matter or mixture which provides some pharmacologic,often beneficial, effect that can be demonstrated in-vivo or in vitro.This includes foods, food supplements, nutrients, nutriceuticals, drugs,vaccines, antibodies, vitamins, and other beneficial agents. As usedherein, these terms further include any physiologically orpharmacologically active substance that produces a localized or systemiceffect in a patient.

[0153] “Pharmaceutically acceptable excipient” or “pharmaceuticallyacceptable carrier” refers to an excipient that can be included in thecompositions of the invention and that causes no significant adversetoxicological effects to the patient.

[0154] “Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a PEG-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in the targettissue. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

[0155] “Multi-functional” in the context of a polymer of the inventionmeans a polymer backbone having 3 or more functional groups containedtherein, where the functional groups may be the same or different, andare typically present on the polymer termini. Multi-functional polymersof the invention will typically contain from about 3-100 functionalgroups, or from 3-50 functional groups, or from 3-25 functional groups,or from 3-15 functional groups, or from 3 to 10 functional groups, orwill contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within thepolymer backbone.

[0156] A “difunctional” polymer means a polymer having two functionalgroups contained therein, typically at the polymer termini. When thefunctional groups are the same, the polymer is said to behomodifunctional. When the functional groups are different, the polymeris said to be heterobifunctional A basic or acidic reactant describedherein includes neutral, charged, and any corresponding salt formsthereof.

[0157] “Polyolefinic alcohol” refers to a polymer comprising an olefinpolymer backbone, such as polyethylene, having multiple pendant hydroxylgroups attached to the polymer backbone. An exemplary polyolefinicalcohol is polyvinyl alcohol.

[0158] As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer may includea minor number of peptide linkages spaced along the repeat monomersubunits, such as, for example, no more than about 1 peptide linkage perabout 50 monomer units.

[0159] The term “patient,” refers to a living organism suffering from orprone to a condition that can be prevented or treated by administrationof a polymer of the invention, typically but not necessarily in the formof a polymer-active agent conjugate, and includes both humans andanimals.

[0160] “Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

[0161] By “residue” is meant the portion of a molecule remaining afterreaction with one or more molecules. For example, a biologically activemolecule residue in the polymer conjugate of the invention is theportion of a biologically active molecule remaining following covalentlinkage to a polymer backbone.

[0162] The term “conjugate” is intended to refer to the entity formed asa result of covalent attachment of a molecule, e.g., a biologicallyactive molecule or any reactive surface, to a reactive polymer molecule,preferably a reactive poly(ethylene glycol).

[0163] The term “electron withdrawing group” refers to a chemical moietythat brings electron density towards itself and away from other areas ofa molecule through either mesomeric mechanisms (i.e., adding or removinglocal electron density through π bonds) or inductive mechanisms (i.e.,an electronegative moiety withdrawing electron density along a σ bond,thereby polarizing the bond).

[0164] The term “steric hindrance” refers to spatial, mechanicalinterference between two chemical groups.

[0165] Stabilized Polymer Maleimides—General Features

[0166] The present invention provides water-soluble and non-peptidicpolymers whose maleimide rings are hydrolytically stable in comparisonto their linkerless maleimide counterparts. Generally, the feature ofresistance to hydrolysis, i.e., with regard to the maleimide ring, isimparted by the introduction of a linker between the polymer segment andthe maleimide. Typically, the linker comprises a saturated acyclic,cyclic, or alicyclic hydrocarbon chain covalently attached to, andimmediately adjacent to, the nitrogen atom of the maleimide. Thestructure and size of the hydrocarbon chain, which may comprise alkylenegroups, cycloalkyl groups, or combinations thereof, in eithersubstituted or non-substituted form, is designed to retard ring openingof the maleimide by i) providing sufficient distance between themaleimide and any electron withdrawing groups in the linker or thepolymer segment, thereby enabling electron release to the maleimidering, and/or ii) providing steric hindrance to the hydrolytic process.In this manner, the linkers described herein stabilize the maleimidering against hydrolysis and resultant ring-opening. Thus, themaleimide-terminated polymers of the invention exhibit greaterstability, e.g., upon synthesis, isolation, and storage, and can beconjugated to biologically active molecules over a wider range of pHvalues without producing significant amounts of open-ring conjugates.Hydrolysis data provided herein is indicative of this point. Thesynthesis of representative stabilized polymer maleimides is describedin Examples I, II, III, IV, V, IX, X, XI, and XII. As can be seen,linear acyclic, branched acyclic, and alicyclic linkers are alleffective in enhancing the stability of the adjacent maleimide ring.Hydrolysis data illustrative of this feature for both the polymerreagents and their corresponding conjugates is provided in Examples VII,VII, and XIII.

[0167] The linkers as described in greater detail below may also includeone or more non-hydrocarbon yet hydrolytically stable and non-reactiveatoms or collection of atoms, such as hydroxyl, sulfur, oxygen, and thelike.

[0168] The maleimide-functionalized polymers of the invention arepreferably hydrolytically stable over a wide pH range, such as fromabout 5 to about 10. In particular, most preferably, the reactivepolymer maleimides of the invention are hydrolytically stable at pHssuitable for conjugation to thiol or amino groups on biologically activemolecules, such as proteins. For example, the polymers of the inventionare preferably resistant to hydrolysis-induced maleimide ring opening(i.e., are not prone to formation of maleimic acid if unconjugated orformation of succinamidic acid if conjugated) at pHs ranging from about7 to about 10, and more preferably at pHs from about 7 to about 8.5. Asdefined herein, a hydrolytically stable maleimide is one in which thehalf-life of the maleimide at 25° C. and a pH of 7.5 in an aqueousmedium (e.g., phosphate buffer) is at least about 16 hours, morepreferably at least about 20 hours, most preferably at least about 28hours.

[0169] The half-life of a polymer maleimide can be determined bymeasuring the concentration of the maleimide-terminated polymer overtime using HPLC or by observing the UV absorption of the maleimide ring.

[0170] The saturated acyclic, cyclic, or alicyclic hydrocarbon linkeradjacent to the maleimide group preferably has a chain length of atleast 3 carbon atoms and contains at least 3 contiguous carbon atoms.More preferably, the linker possesses at least about 4 carbon atoms,most preferably at least about 5 or 6 carbon atoms. The chain length ismeasured as the number of carbon atoms forming the shortest atom chainlinking the nitrogen atom of the maleimide to the polymer segment.Typically, the total number of carbon atoms in the linker includingchain substituents, ranges from 4 to about 20 atoms, preferably 4 toabout 12 atoms, more preferably 4 to about 10 atoms and most preferably5 to about 8 atoms. The invention includes linkers having, for example,4, 5, 6, 7, 8, 9, 10, 11, and 12 total carbon atoms.

[0171] General Structural Features of the Polymer Maleimide

[0172] Generally speaking, the reactive polymers of the inventionpossesses a water soluble polymer segment that is connected to amaleimide ring via a hydrolytically stable linker. The hydrolyticallystable linker is effective to impart hydrolytic stability to themaleimide ring to which it is directly covalently attached. Moreparticularly, the polymer segment, referred to herein generally as POLY,is covalently attached to the hydrolytically stable linker, X, via anintervening —O—, —C(O)—NH—, or O—C(O)—NH— group. X typically contains atleast 3 contiguous saturated carbon atoms. Preferably although notnecessarily, the resultant polymer maleimide is absent aromatic groupsand ester linkages.

[0173] Also provided herein are polymers having both the generalized andspecific illustrative structural features described for the stabilizedpolymer maleimides, with the exception that the maleimide ring isreplaced by an amino group, preferably a primary amino group. Thus, allstructures and descriptions herein directed towards maleimide-terminatedpolymer reagents should be extended to their amino-terminatedcounterparts as described above.

[0174] The linker, X, typically contains from about 1 to about 20 carbonatoms. Generally, X is a hydrocarbon chain possessing only carbon andhydrogen atoms, however, in certain embodiments, X may containadditional non-reactive atoms or functional groups such as hydroxylgroups, ethers, thioethers, or other non-reactive groups. Preferably,such groups or atoms are positioned remote from the maleimide ring. Morepreferably, such non-reactive atoms or groups are positioned a distanceof at least 4 carbons from the maleimide nitrogen. Even more preferably,such additional non-reactive atoms or groups contained in X arepositioned at least 4 carbons, at least 5 carbons or at least 6 carbonsor more distant from the maleimide nitrogen. Such groups are more likelyto be contained within cycloalkyl or alicyclic X's than in their acycliccounterparts, simply due to their presence in many commerciallyavailable starting materials.

[0175] A polymer maleimide of the invention is generally characterizedby the following formula:

[0176] where b is 0 or 1. In structure I, when both the carbonyl and —NHgroups are absent (i.e., each subscript is equal to zero), b is equalto 1. Features of the linker X are described and exemplified in greaterdetail in the section that follows.

[0177] The Linker Moiety

[0178] As mentioned previously, the linker, X, comprises a saturatedacyclic or cyclic or alicyclic hydrocarbon moiety adjacent to thenitrogen atom of the maleimide ring. The size and structure of X isdesigned to improve the hydrolytic stability of the maleimide ring,generally by increasing the distance between the maleimide ring andelectron withdrawing groups present in the molecule or by providingsteric hindrance to the maleimide hydrolysis reaction. Typically, Xcontains a total of about 3 to about 20 carbon atoms. More particularly,X can possess a total number of carbon atoms selected from the groupconsisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, and 20. Preferred ranges for total number of carbon atoms in thelinker X are from about 3 to about 20, or from about 4 to about 12, orfrom about 4 to about 10, or from about 5 to about 8 atoms.

[0179] Exemplary hydrocarbon linkages include straight chain saturatedacyclic hydrocarbons comprising at least 3 contiguous carbon atoms, suchas trimethylene, tetramethylene, pentamethylene, and hexamethylene, andso forth. That is to say, in its simplest form, X equals —(CH₂)_(y),where y ranges from 3 to about 20. That is to say, Y may possess any ofthe following values: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20. Alternatively, X can be branched, i.e., can containone or even two substituents, at any one or more of the carbon positionsin the chain. That is to say, X can be branched at the carbon α to themaleimidyl group, or at the carbon β to the maleimidyl group, or at thecarbon γ to the maleimidyl group. For hydrocarbon chains having up to 19carbon atoms, any one of positions 1 to 19 (with position 1 being theone proximal to the maleimide ring) may be branched. For instance, foran exemplary saturated hydrocarbon chain having from 2 to 19 carbonatoms designatedC₁-C₂-C₃-C₄-C₅-C₆-C₇-C₈-C₉-C₁₀-C₁₁-C₁₂-C₁₃-C₁₄-C₁₅-C₁₆-C₁₇-C₁₈-C₁₉-, anyone or more of carbons C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, or C₁₉, depending on the total numberof carbons in the chain, may be branched, that is to say, may possessone or even two substituents. Typically, a branching group is an alkylgroup, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, or substituted cycloalkyl. Particularly preferredcycloalkyls are cyclopentyl, cyclohexyl, cycloheptyl, and the like, andany of the previous cycloalkyls having one or more methylene groups(e.g., methylene, dimethylene, trimethylene, tetramethylene, etc.)connecting the cycloalkyl ring to the branching carbon. Preferably, inany given saturated hydrocarbon chain or alicyclic linker, 4 or fewercarbon atoms are branched, with the overall number of branchingpositions preferably equal to 1, 2, 3, or 4. Embodiments wherein the“branch” points taken together form a saturated ring or ring system(e.g., bicyclic, tricyclic, etc.) are discussed separately below. Mostpreferably, when X is branched, the branching carbon is singly branched,i.e., has one rather than two branching substituents.

[0180] For example, the linkage may possess the structure —(CR₁R₂)_(y)—,wherein R₁ and R₂ are each independently H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl, and y is an integer from about 1 to about 20,preferably from about 3 to about 20, and even more preferably from 4 toabout 12. When X is branched, preferably the branching is at one or moreof C1, C1, C3, or C4, that is to say, the carbon atom positions closestto the maleimide ring, in order to provide maximal steric hindrance tothe maleimide ring hydrolysis reaction. (When discussing carbon atompositions within the linker, X, C1 refers to the carbon atom adjacent tothe maleimide nitrogen, and so on). When X is branched, the branchinggroups (e.g., alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl)typically contain fewer than 8 or so carbon atoms. When the branchinggroup is alkyl or cycloalkyl, preferably the alkyl group is lower alkylsuch as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, penyl,cyclopentyl, hexyl, and cyclohexyl.

[0181] As dicussed above, the linker X itself can be cyclic oralicyclic. Specifically, X may possess the form:

[0182] where CYC_(a) is a cycloalkylene group having “a” ring carbons,where the value of “a” ranges from 3 to 12; p and q are eachindependently 0 to 20, and p+q+a≦20, R¹, in each occurrence, isindependently H or an organic radical that is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, andR², in each occurrence, is independently H or an organic radical that isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl. In a preferred embodiment, R₁ and R₂, in eachoccurrence, are both H. Preferably, p and q each independently are 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. Even more preferably, p and q eachindependently range from 0 to 6 or 0 to 4. Preferably, the cycloalkylring represented by CYC_(a) contains from 5 to about 12 ring carbonatoms, and even more preferably from about 6 to about 10 ring carbonatoms. Representative cycloalkyl groups include C3-C8 cycloalkylene,such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene,and cycloheptylene, and cyclooctylene. CYC_(a) may optionally besubstituted with one or more alkyl groups, preferably lower alkylgroups, which can be at any position within the ring. In instances whereCYC_(a) is substituted with only two substituents, i.e., (CR₁R₂)_(p) and(CR₁R₂)_(q) as shown in structure IV above, the substituents cansimilarly be positioned on any one or more carbons in the ring. Forinstance, for a cyclopenylene, the substituents can be at either the1,1-, 1,2-, or 1,3-positions. For a cyclohexylene ring, the substituentscan be at the 1,1-, 1,2-, 1,3-, or 1,4-positions. Particularly preferredembodiments are those where R₁ and R₂ are H in all instances, and p andq are each independently selected from 0, 1, 2, and 3. Illustrativelinkers include 1,2-(CH₂)_(0,1,2,3)—C₆H₄—(CH₂)_(0,1,2,3), and1,3-(CH₂)_(0,1,2,3)—C₆H₄—(CH₂)_(0,1,2,3), and1,4-(CH₂)_(0,1,2,3)—C₆H₄—(CH₂)_(0,1,2,3)—. In instances in which CYC_(a)possesses only two substituents, the substituents may be either cis ortrans. When CYC_(a) contains more than two substituents, thesubstituents may be in any relative orientation to one another.

[0183] CYC_(a) also encompasses bicyclic rings. Exemplary bicyclic ringscorresponding to CYC_(a) include rings such as bicyclo[1.1.1]pentane,bicyclo[2.2.1]hexane, bicyclo[2.2.1]heptane, bicycle[2.2.2]octane,bicyclo[3.1.0]hexane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane,bicyclo[3.3.1]nonane, bicyclo[3.3.2]decane, bicyclo[3.3.3]undecane andthe like. CYC also encompasses tricyclic ring systems such asadamantane. Some of these bi- and tricyclic systems are shown below:

[0184] These rings may possess alkylene or substituted alkylene groupscorresponding to —[C(R₁)(R₂)]_(p) and —[C(R₁)(R₂)]_(q) in any availableposition within the ring system. Moreoever, the bicyclic or tricyclicring may also possess additional substituents in addition to—[C(R₁)(R₂)]_(p) and —[C(R₁)(R₂)]_(q). Preferably, such substituents arelower alkyl, hydroxyl, sulfhydryl, or halide. Representative polymermaleimides having bi- and tricyclic ring systems are provided in FIGS.1A and 1B.

[0185] The linkage, L, in structure I may further include anon-hydrocarbon portion adjacent to the polymer segment andinterconnected to X as described above. Exemplary non-hydrocarbonportions adjacent to the polymer segment include —O—, —O—C(O)—NH—,—C(O)—NH—, —CH₂—C(O)—NH—, —NH—C(O)—O—, NH—C(O)—NH—, —NH—, and —S—, andare preferably hydrolytically stable. Particularly preferrednon-hydrocarbon portions include —O—, —O—C(O)—NH—, —C(O)—NH—. In a lesspreferred embodiment, the nitrogen amide of the amide or carbamatefunction is a tertiary nitrogen, e.g., having a methyl or ethyl orsimilar group in place of the hydrogen as shown.

[0186] Exemplary linkages including a hydrocarbon chain according to thepresent invention are shown in Table 1 below. TABLE 1 Exemplary linkersfor Maleimide-Terminated Polymers

L₁ =

Designation P L1-AMDE ethylene, —(CH₂)₂— L1-AMPE pentamethylene,—(CH₂)₅— L1-MCH

L1-TEPE

L₂ = —NH—Q— Designation Q L2-TEME tetramethylene, —(CH₂)₄— L2-ILEXAhexamethylene, —(CH₂)₆— L2-EPEN

L₃ = —O—Z Designation Z L3-ET ethylene, —(CH₂)₂— L3-TME trimethylene,—(CH₂)₃— L3-TEME tetramethylene, —(CH₂)₄— L3-PENT pentamethylene,—(CH₂)₅— L3-HEXA hexamethylene, —(CH₂)₆— L₄ = —CH₂—W— Designation WL4-PAHE —C(O)NH—(CH₂)₆— L4-BAHE —CH₂—C(O)NH—(CH₂)₆— L4-TMPA

L4-ETPA

L4-HEDA

L4-CMEN

L₅ =

Designation V L5-TMPE

L5-HEXA —(CH₂)₆—

[0187] Many of the linkages in Table 1 are effective to retardhydrolysis of the maleimide ring, although some are more effective thanothers. Several linkages in Table 1 provide steric hindrance to attackof the maleimide ring nitrogen by water, making the maleimide resistantto hydrolysis. These linkers include those comprising L4-TMPA, L4-CMEN,L5-TMPE, L1-TEPE, L2-EPEN, L4-ETPA and L4-HEDA. Linkers comprisingL4-TMPA and L5-TMPE, which are based on the readily availablecorresponding symmetrical tertiary diamine, and L4-CMEN, which is basedon the commercially available diamine derivative of naturally occurringp-menthane, are exemplary linkages that reduce the rate of hydrolysis ofthe maleimide ring by providing both steric hindrance and adequatespacing between the ring and electron withdrawing groups. Particularlypreferred are linkers containing a cyclohexylene ring, as can be seenfrom the hydrolysis data provided in Table 4. As can be seen from thehydrolysis data, the 1,3-dimethylene-cyclohexylene linker imparts to theresultant PEG-maleimide a particular stability towards hydrolysis. Infact, its hydrolysis half life is 8 times longer than that of itslinkerless maleimide counterpart. The 1,4-dimethylene-cyclohexylenelinker also results in a stable maleimide polymer having a hydrolysishalf life that is over two and half times longer that that of itslinkerless maleimide counterpart.

[0188] The Polymer Segment

[0189] As shown in the illustrative structures above, a maleimideterminated polymer of the invention contains a water-soluble polymersegment. Representative POLYs include poly(alkylene glycols) such aspoly(ethylene glycol), poly(propylene glycol) (“PPG”), copolymers ofethylene glycol and propylene glycol, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline, andpoly(N-acryloylmorpholine). POLY can be a homopolymer, an alternatingcopolymer, a random copolymer, a block copolymer, an alternatingtripolymer, a random tripolymer, or a block tripolymer of any of theabove. The water-soluble polymer segment is preferably, although notnecessarily, a poly(ethylene glycol) “PEG” or a derivative thereof.

[0190] The polymer segment can have any of a number of differentgeometries, for example, POLY can be linear, branched, or forked. Mosttypically, POLY is linear or is branched, for example, having 2 polymerarms. Although much of the discussion herein is focused upon PEG as anillustrative POLY, the discussion and structures presented herein can bereadily extended to encompass any of the water-soluble polymer segmentsdescribed above.

[0191] Any water-soluble polymer having at least one reactive terminuscan be used to prepare a polymer maleimide in accordance with theinvention and the invention is not limited in this regard. Althoughwater-soluble polymers bearing only a single reactive terminus can beused, polymers bearing two, three, four, five, six, seven, eight, nine,ten, eleven, twelve or more reactive termini suitable for conversion toa stabilized polymer maleimide as set forth herein can be used.Advantageously, as the number of hydroxyl or other reactive moieties onthe water-polymer segment increases, the number of available sites forintroducing a Tinkered maleimido group increases. Nonlimiting examplesof the upper limit of the number of hydroxyl and/or reactive moietiesassociated with the water-soluble polymer segment include from about 1to about 500, from 1 to about 100, from about 1 to about 80, from about1 to about 40, from about 1 to about 20, and from about 1 to about 10.

[0192] In turning now to the preferred POLY, PEG encompassespoly(ethylene glycol) in any of its linear, branched or multi-arm forms,including end-capped PEG, forked PEG, branched PEG, pendant PEG, andless preferably, PEG containing one or more degradable linkageseparating the monomer subunits, to be more fully described below. Inone embodiment of the invention, the polymer segment is absent an esterlinkage.

[0193] A PEG polymer segment comprises the following:—(CH₂CH₂O)_(n)—CH₂CH₂—, where (n) typically ranges from about 3 to about4,000, or from about 3 to about 3,000, or more preferably from about 20to about 1,000.

[0194] POLY can also be end-capped, for example an end-capped PEG wherePEG is terminally capped with an inert end-capping group. Preferredend-capped PEGs are those having as an end-capping moiety such asalkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy,alkynyloxy, substituted alkynyloxy, aryloxy, substituted aryloxy.Preferred end-capping groups are methoxy, ethoxy, and benzyloxy. Theend-capping group can also advantageously comprise a phospholipid,although the polymer may also be absent a lipid. Exemplary phospholipidsinclude phosphatidylcholines, such as dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine,disteroylphosphatidylcholine, behenoylphosphatidylcholine,arachidoylphosphatidylcholine, and lecithin.

[0195] Referring now to any of the structures containing a polymersegment, POLY, POLY may correspond or comprise the following:

[0196] “Z-(CH₂CH₂O)_(n)—” or “Z-(CH₂CH₂O)_(n)—CH₂CH₂—”,

[0197] where n ranges from about 3 to about 4000, or from about 10 toabout 4000, and Z is or includes a functional group, which may be areactive group or an end-capping group. Examples of Z include hydroxy,amino, ester, carbonate, aldehyde, acetal, aldehyde hydrate, ketone,ketal, ketone hydrate, alkenyl, acrylate, methacrylate, acrylamide,sulfone, thiol, carboxylic acid, isocyanate, isothiocyanate, hydrazide,urea, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, alkoxy, benzyloxy, silane, lipid, phospholipid, biotin,and fluorescein, including activated and protected forms thereof whereapplicable. Preferred are functional groups such asN-hydroxysuccinimidyl ester, benzotriazolyl carbonate, amine,vinylsulfone, maleimide, N-succinimidyl carbonate, hydrazide,succinimidyl propionate, succinimidyl butanoate, succinimidyl succinate,succinimidyl ester, glycidyl ether, oxycarbonylimidazole, p-nitrophenylcarbonate, aldehyde, orthopyridyl-disulfide, and acrylol.

[0198] These and other functional groups, Z, are described in thefollowing references, all of which are incorporated by reference herein:N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol.Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461).

[0199] Again, the POLY structures shown immediately above may representlinear polymer segments, or may form part of a branched or forkedpolymer segment. In an instance where the polymer segment is branched,the POLY structures immediately above may, for example, correspond tothe polymer arms forming part of the overall POLY structure.Alternatively, in an instance where POLY possesses a forked structure,the above POLY structure may, for example, correspond to the linearportion of the polymer segment prior to the branch point.

[0200] POLY may also correspond to a branched PEG molecule having 2arms, 3 arms, 4 arms, 5 arms, 6 arms, 7 arms, 8 arms or more. Branchedpolymers used to prepare the polymer maleimides of the invention maypossess anywhere from 2 to 300 or so reactive termini. Preferred arebranched polymer segments having 2 or 3 polymer arms. An illustrativebranched POLY, as described in U.S. Pat. No. 5,932,462, corresponds tothe structure:

[0201] In this representation, R″ is a nonreactive moiety, such as H,methyl or a PEG, and P and Q are nonreactive linkages. In a preferredembodiment, the branched PEG polymer segment is methoxy poly(ethyleneglycol) disubstituted lysine.

[0202] In the above particular branched configuration, the branchedpolymer segment possesses a single reactive site extending from the “C”branch point for positioning of the reactive maleimide group via alinker as described herein. Branched PEGs such as these for use in thepresent invention will typically have fewer than 4 PEG arms, and morepreferably, will have 2 or 3 PEG arms. Such branched PEGs offer theadvantage of having a single reactive site, coupled with a larger, moredense polymer cloud than their linear PEG counterparts.

[0203] One particular type of branched PEG maleimide corresponds to thestructure: (MeO-PEG-)_(i)G-[O]_(b)—C(O)—NH—X-MAL, where MAL representsmaleimide, i equals 2 or 3, and G is a lysine or other suitable aminoacid residue.

[0204] An illustrative branched polymer maleimide of the invention hasthe structure shown below, where X is any of the herein describedhydrolytically stable linkers.

[0205] The synthesis of a polymer of the invention having the structuralfeatures embodied in XVIII above is provided in Example 1. Branched PEGsfor use in preparing a polymer maleimide of the invention additionallyinclude those represented more generally by the formula R(PEG)_(n),where R is a central or core molecule from which extends 2 or more PEGarms. The variable n represents the number of PEG arms, where each ofthe polymer arms can independently be end-capped or alternatively,possess a reactive functional group at its terminus, such as a maleimideor other reactive functional group. In such multi-armed embodiments ofthe invention, each PEG arm typically possesses a maleimide group at itsterminus. Branched PEGs such as those represented generally by theformula, R(PEG)_(d), above possess 2 polymer arms to about 300 polymerarms (i.e., n ranges from 2 to about 300). Branched PEGs such as thesepreferably possess from 2 to about 25 polymer arms, more preferably from2 to about 20 polymer arms, and even more preferably from 2 to about 15polymer arms or fewer. Most preferred are multi-armed polymers having 3,4, 5, 6, 7 or 8 arms.

[0206] Preferred core molecules in branched PEGs as described above arepolyols. Such polyols include aliphatic polyols having from 1 to 10carbon atoms and from 1 to 10 hydroxyl groups, including ethyleneglycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkylcycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose,galactose, glucose, fructose, sorbose, mannose, pyranose, altrose,talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.Additional aliphatic polyols include derivatives of glyceraldehyde,glucose, ribose, mannose, galactose, and related stereoisomers. Othercore polyols that may be used include crown ether, cyclodextrins,dextrins and other carbohydrates such as starches and amylose. Preferredpolyols include glycerol, pentaerythritol, sorbitol, andtrimethylolpropane.

[0207] A representative multi-arm polymer structure of the typedescribed above is:

[0208] where d is an integer from 3 to about 100, and R is a residue ofa central core molecule having 3 or more hydroxyl groups, amino groups,or combinations thereof.

[0209] Multi-armed PEGs for use in preparing a polymer maleimide of theinvention include multi-arm PEGs available from Nektar, Huntsville, Ala.In a preferred embodiment, a multi-armed polymer maleimide of theinvention corresponds to the following, where the specifics of theTinkered maleimide portion of the molecule are provided elsewhereherein.

[0210] where

[0211] PEG is —(CH₂CH₂O)_(n)CH₂CH₂—,

[0212] M is:

[0213] and m is selected from the group consisting of 3, 4, 5, 6, 7, and8.

[0214] Alternatively, the polymer maleimide may possess an overallforked structure. An example of a forked PEG corresponds to thestructure:

[0215] where PEG is any of the forms of PEG described herein, A is alinking group, preferably a hydrolytically stable linkage such asoxygen, sulfur, or —C(O)—NH—, F and F′ are hydrolytically stable spacergroups that are optionally present, and the other variablescorresponding to the hydrolytically stable linker, X, and maleimide(MAL) portion are as defined above. Both the general and specificdescriptions of possible values for X are applicable to the embodimentabove, structure XVIII. Examplary linkers and spacer groupscorresponding to A, F and F′ are described in International ApplicationNo. PCT/US99/05333, and are useful in forming polymer segments of thistype for use in the present invention. F and F′ are spacer groups thatmay be the same of different. In one particular embodiment of the above,PEG is MPEG, A corresponds to —C(O)—NH—, and F and F′ are both methyleneor —CH₂—. This type of polymer segment is useful for reaction with twoactive agents, where the two active agents are positioned a precise orpredetermined distance apart, depending upon the selection of F and F′.

[0216] Another version of a polymer reagent of the invention having aforked polymer segment corresponds to:

[0217] where the variables are as described above. Preferably, X in thisembodiment is a saturated cyclic or alicyclic hydrocarbon chain having atotal of 3 to about 20 carbon atoms.

[0218] An exemplary branched PEG corresponding to “PEG” in the aboveformula is mPEG disubstituted lysine, where “PEG” corresponds to:

[0219] Alternatively, the PEG polymer segment for use in preparing apolymer maleimide of the invention may be a PEG molecule having pendantreactive groups along the length of the PEG chain rather than at theend(s), to yield a stabilized polymer maleimide having one or morependant maleimide groups attached to the PEG chain by a linker, X.

[0220] Further, in a less preferred embodiment, the polymer segmentitself may possess one or more weak or degradable linkages that aresubject to hydrolysis. Illustrative degradable linkages that may bepresent in the polymer segment include but are not limited to carbonate,imine, phosphate ester, and hydrazone.

[0221] Generally, the nominal average molecular mass of thewater-soluble polymer segment, POLY will vary. The nominal averagemolecular mass of POLY typically falls in one or more of the followingranges: about 100 daltons to about 100,000 daltons; from about 500daltons to about 80,000 daltons; from about 1,000 daltons to about50,000 daltons; from about 2,000 daltons to about 25,000 daltons; fromabout 5,000 daltons to about 20,000 daltons. Exemplary nominal averagemolecular masses for the water-soluble polymer segment POLY includeabout 1,000 daltons, about 5,000 daltons, about 10,000 daltons, about15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000daltons, and about 40,000 daltons. Low molecular weight POLYs possessmolecular masses of about 250, 500, 750, 1000, 2000, or 5000 daltons.

[0222] Polymer Amines

[0223] The present invention also extends to amine counterparts of anyand all of the above-described structures, with the exception that theheretofore described maleimide ring is replaced with an amino group,preferably —NH₂.

[0224] More particularly, the invention extends to a water-solublepolymer having the structure:

[0225] where POLY, b and X are as described above.

[0226] Representative polymer amines include those having thegeneralized structures shown below:

[0227] wherein q and p each independently range from 0 to 6, and thesubstituents on said cyclohexylene ring are either cis or trans.

[0228] wherein q and p each independently range from 0 to 6, and thesubstituents on said cyclohexylene ring are either cis or trans.

[0229] The amine-terminated polymers represented generally by VIII havea number of uses to be described in greater detail below. For instance,they can be converted to the corresponding maleimide-terminated polymersof the invention, or alternatively, used without further modificationfor covalent attachment to active agents or surfaces, or for forminghydrogels.

[0230] Hyrolytic Stability and Methods of Preparation

[0231] As described above, the polymer maleimides provided herein areresistant to hydrolysis, as demonstrated for both the polymer reagentsthemselves (Examples VII and XIII) and for their correspondingconjugates (Example VIII). A polymer maleimide of the invention has ahydrolysis half-life that is longer than that of its linkerless polymermaleimide counterpart, when measured under the same conditions. That isto say, a Tinkered polymer maleimide of the invention possesses a rateof hydrolysis that is slower than that of its corresponding linkerlessversion, meaning that the maleimide ring remains intact longer underessentially the same conditions. For instance, in looking at thehydrolysis data in Examples 7 and 14, Tables 2 and 4, each of therepresentative linkers has a hydrolysis half-life that is extended overthat of the linkerless maleimide (“3-ET”).

[0232] The polymer maleimides of the invention can be prepared by anumber of alternative routes including the following. In one approach, amaleimide-terminated polymer of the invention is prepared by reacting afunctional group attached to a polymer segment (i.e., an activatedpolymer segment) with a functional group attached to a bifunctionallinker. Reacting the polymer segment with a bifunctional linker reagentresults in covalent attachment, through a hydrolytically stable linkage,of the linker to the polymer segment. The remaining functional group onthe bifunctional linker reagent is either a maleimide or a functionalgroup that can be readily converted to a maleimide.

[0233] For example, the linker reagent may possess the structure A-L-B,wherein A is a first functional group that is reactive with a secondfunctional group on the polymer segment to thereby form a hydrolyticallystable linkage, L, to form POLY-L-B, where B is a maleimide or afunctional group that can be readily converted to a maleimide (e.g., anamine that can be converted to a maleimide by reaction withmethoxycarbonylmaleimide). In the above approach, A can be any of anumber of functional groups such as halo, hydroxyl, active ester such asN-succinimidyl ester, active carbonate, acetal, aldehyde, aldehydehydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone,thiol, carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, and epoxide.Particular xamples of linker reagents include 1,4-dibromobutane,1,5-dibromopentane, 1,6-dibromohexane, N-succinimidylε-maleimidohexanoate), N-succinimidyl(ε-maleimidopentanoate),N-(γ-maleimidobutryloxy)succinimide ester,N-(γ-maleimidocaproyloxy)succinimide ester,4,7,10-trioxa-1,3-tridecanediamine,4-(maleimidomethyl)-1-cyclohexanecarboxylic acid—NHS ester,tetramethylenediamine, pentamethylenediamine, hexamethylenediamine,2,5-diamino-2,5-dimethylhexane, 1,3-cyclohexylbis(methylamine), and1,4-cyclohexylbis(methylamine). Such linker reagents are eithercommercially available, for example from Pierce Chemical Company, or canbe prepared from commercially available starting materials usingmethodology known in the art.

[0234] This approach is shown more particularly as method 1 below, as itrelates to the formation of polymer malemides containing an amide orurethane bond connecting the polymer segment to the linker, X.

[0235] Method 1.

[0236] In a method similar to the above, designated method 2, thereactive polymer starting material, POLY-[O]_(b)—C(O)-LG, is reactedwith a diamine reagent, H₂N—X—NH₂, to form the corresponding polymeramine intermediate, POLY-[O]_(b)—C(O)—HN—X—NH. This intermediate is thenconverted to the corresponding stabilized maleimide-terminated polymer.This method is advantageous in that many diamine reagents suitable forforming a stabilized polymer maleimide as described herein arecommercially available. Moreover, polymer-amine intermediates can bemore easily purified than their maleimide counterparts, e.g., by columnchromatography, to thereby provide a polymer maleimide product that issignificantly absent other undesirable polymer-derived side-products,such as PEG-diol and PEG-diol derived impurities. Method 2 is shownbelow.

[0237] Method 2.

[0238] In methods 1 and 2, LG represents a leaving group, while theother variables are as described previously. The reactive polymerstarting material, POLY-[O]_(b)—C(O)-LG, may be, for example, an acylhalide, a haloformate, an anhydride, or an active ester. Leaving groupsuseful in the methods include halides (e.g., chloro, bromo, and iodo),N-hydroxysuccinimide, N-hydroxybenzotriazole, and para-nitrophenolate.The coupling reaction to form the amide or urethane bond is generallycarried out in a dry organic solvent, preferably under an inertatmosphere such as nitrogen or argon. Suitable solvents includeacetonitrile, chlorinated hydrocarbons such as chloroform anddichloromethane, aromatic hydrocarbons such as benzene, toluene, andxylene, and solvents such as acetone, and tetrahydrofuran. The reactionis typically carried out at temperatures ranging from about 0 to 100°C., depending upon the type of solvent employed and the reactivity ofthe particular reagents themselves. The coupling is generally conductedin the presence of a base. Bases include trialkylamines such as triethylamine, pyridine, 4-(dimethylamino)pyridine, and inorganic bases such assodium carbonate.

[0239] Representative bicyclic and tricyclic diamine reactantscorresponding to H₂N—X—NH₂ in method 2 above are provided in FIG. 2. Inmethod 2, it may be necessary in certain instances to protect one of theamino groups in H₂N—X—NH₂ using, for example, a conventionalamino-protecting group such as t-BOC or FMOC. The protecting group inPOLY-[O]_(b)—C(O)—HN—X—NH is then typically removed prior to furtherpurification or transformation. See for example, Examples 10 and 11. Ininstances where purification of the intermediate polymer amine isundertaken, any of a number of purification approaches can be used suchas precipitation or chromatography, although preferred is ion exchangechromatography due to the presence of an amino group on the intermediatepolymer amine.

[0240] In continuing with the approach in method 2, the intermediatepolymer amine is then converted to the corresponding maleimide.Generally, this conversion is carried out by reactingPOLY-[O]_(b)—C(O)—H₂N—X—NH₂ with a reagent such asN-methoxycarbonylmaleimide,exo-7-oxa[2.2.1]bicycloheptane-2,3-dicarboxylic anhydride, or maleicanhydride, under conditions suitable for formingPOLY-[O]_(b)—C(O)—H₂N—X-MAL.

[0241] A preferred reagent is N-methoxycarbonylmaleimide, and in thisinstance, the conversion to the maleimide is carried out in water or anaqueous mixture of water and a water miscible solvent such asacetonitrile or acetone. The conversion reaction is generally carriedout at temperatures ranging from about 0 to 80° C., at pHs ranging fromabout 6.5 to 9

[0242] When the reagent is maleic anhydride, POLY-[O]_(b)—C(O)—H₂N—X—NH₂is reacted with maleic anhydride under conditions effective to formPOLY-[O]_(b)—C(O)—NH—X—NH—C(O)CH═CHCOOH (XI) as an intermediate. Thisintermediate is then heated under conditions effective to promotecyclization by elimination of water to form POLY-[O]_(b)—C(O)—NH—X-MAL.The efficiency of the cyclization reaction to form the maleimide ringtypically ranges from about 15 to about 80 percent.

[0243] Generally, the product, POLY-[O]_(b)—C(O)—H₂N—X-MAL is recoveredfrom the reaction mixture, and optionally further purified. In instanceswhere the product is formed by method 2, further purification, forexample, to remove polymer-derived impurities, may be unnecessary ifpurification is carried out on the amine-precursor, for example, by ionexchange chromatography. Preferably, the recovered product,POLY-[O]_(b)—C(O)—H₂N—X-MAL has a polymer purity of greater than about80%.

[0244] Examples 1 and 5 illustrate a method of forming a reactivepolymer of the invention using a linker that comprises a terminalmaleimide group. In Example 1, a polymer segment modified to contain areactive amino group is reacted with bifunctional linker comprising anactivated ester and a maleimide group. A similar approach is utilized inExample 5 to prepare a polymer maleimide having a cyclohexylenecontaining linker. Examples 2, 3, and 4 demonstrate formation of apolymer amine intermediate that is then converted to the correspondingmaleimide. Examples 9, 10, 11, and 12 demonstrate the synthesis ofcycloalkylene-containing linkers by method 2 above, where a reactivepolymer starting material, POLY-[O]_(b)—C(O)-LG, is reacted with adiamine reagent to form POLY-[O]_(b)—C(O)—H₂N—X—NH₂, which is thenconverted to the corresponding maleimide-terminated product.

[0245] Storage of Polymer Maleimide Reagents

[0246] Preferably, the polymer maleimides of the invention, as well astheir amino counterparts, are stored under an inert atmosphere, such asunder argon or under nitrogen. Due to the potential of the maleimideportion of the molecule for reaction with water (e.g., by exposure tomoisture to form the corresponding ring-opened form), it is alsopreferable to minimize exposure of the polymer maleimides of theinvention to moisture. Thus, preferred storage conditions are under dryargon or another dry inert gas at temperatures below about −15° C.Storage under low temperature conditions is preferred, since rates ofundesirable side reactions, such as maleimide ring opeing, are slowed atlower temperatures. In instances where the polymer segment of thepolymer product is PEG, the PEG portion can react slowly with oxygen toform peroxides along the PEG portion of the molecule. Formation ofperoxides can ultimately lead to chain cleavage, thus increasing thepolydispersity of the PEG reagents provided herein. In view of theabove, it is additionally preferred to store the PEG maleimides andrelated polymers of the invention in the dark.

[0247] Biologically Active Conjugates

[0248] Coupling Chemistry, Separation, Storage

[0249] The Conjugates

[0250] The present invention also encompasses conjugates formed byreaction of any of the herein described stabilized polymer maleimides ortheir corresponding polymer amine counterparts. In particular, theherein-described polymer maleimides are useful for conjugation to activeagents or surfaces bearing at least one thiol or amino group availablefor reaction, while the herein-described polymer amines are useful forconjugation to active agents or surfaces bearing at least one carboxylicgroup available for reaction.

[0251] For instance, a conjugate of the invention may possess thefollowing structure:

[0252] where “—S-active agent” represents an active agent, preferably abiologically active agent, comprising a thiol (—SH) group, and the othervariables are as described previously. In instances where the activeagent is a biologically active agent or small molecule containing onlyone reactive thiol group, the resulting composition may advantageouslycontain only a single polymer conjugate species, due to the relativelylow number of sulfhydryl groups typically contained within a protein andaccessible for conjugation. In some instances, a protein or smallmolecule or other active agent is engineered to possess a thiol group ina known position, and will similarly result in a composition comprisingonly a single polymer conjugate species.

[0253] Alternatively, a conjugate of the invention may possess thefollowing structure:

[0254] In structure XV, “—NH-active agent” represents an active agent orsurface comprising an amino group, preferably a biologically activeagent, and the other variables are as previously described.

[0255] The polymer amines of the invention, when used directly, can beused to provide conjugates of the following type:

[0256] The polymer conjugates provided herein, particularly thosederived from the stabilized polymer maleimides of the invention,similarly possess the feature of improved hydrolytic stability tomaleimide ring opening. This feature is demonstrated in Example VIII.The synthesis of exemplary conjugates, using both the model compound,2-mercaptoethanol, and an illustrative protein, is described in Examples6, 14, 15, 16, and 17.

[0257] Methods of Conjugation

[0258] Suitable conjugation conditions are those conditions of time,temperature, pH, reagent concentration, solvent, and the like sufficientto effect conjugation between a polymeric reagent and an active agent.As is known in the art, the specific conditions depend upon, among otherthings, the active agent, the type of conjugation desired, the presenceof other materials in the reaction mixture and so forth. Sufficientconditions for effecting conjugation in any particular case can bedetermined by one of ordinary skill in the art upon a reading of thedisclosure herein, reference to the relevant literature, and/or throughroutine experimentation.

[0259] Exemplary conjugation conditions include carrying out theconjugation reaction at a pH of from about 6 to about 10, and at, forexample, a pH of about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.The reaction is allowed to proceed from about 5 minutes to about 72hours, preferably from about 30 minutes to about 48 hours, and morepreferably from about 4 hours to about 24 hours or less. Temperaturesfor conjugation reactions are typically, although not necessarily, inthe range of from about 0° C. to about 40° C.; conjugation is oftencarried out at room temperature or less. Conjugation reactions are oftencarried out in a buffer such as a phosphate or acetate buffer or similarsystem.

[0260] With respect to reagent concentration, an excess of the polymericreagent is typically combined with the active agent. In some cases,however, it is preferred to have stoichiometic amounts of the number ofreactive groups on the polymeric reagent to the amount of active agent.Exemplary ratios of polymeric reagent to active agent include molarratios of about 1:1 (polymeric reagent:active agent), 1.5:1, 2:1, 3:1,4:1, 5:1, 6:1, 8:1, or 10:1. The conjugation reaction is allowed toproceed until substantially no further conjugation occurs, which cangenerally be determined by monitoring the progress of the reaction overtime.

[0261] Progress of the reaction can be monitored by withdrawing aliquotsfrom the reaction mixture at various time points and analyzing thereaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any othersuitable analytical method. Once a plateau is reached with respect tothe amount of conjugate formed or the amount of unconjugated polymerremaining, the reaction is assumed to be complete. Typically, theconjugation reaction takes anywhere from minutes to several hours (e.g.,from 5 minutes to 24 hours or more). The resulting product mixture ispreferably, but not necessarily purified, to separate out excessreagents, unconjugated reactants (e.g., active agent) undesiredmulti-conjugated species, and free or unreacted polymer. The resultingconjugates can then be further characterized using analytical methodssuch as MALDI, capillary electrophoresis, gel electrophoresis, and/orchromatography.

[0262] More preferably, a polymer maleimide of the invention istypically conjugated to a sulfhydryl-containing active agent at pHsranging from about 6-9 (e.g., at 6, 6.5, 7, 7.5, 8, 8.5, or 9), morepreferably at pHs from about 7-9, and even more preferably at pHs fromabout 7 to 8. Generally, a slight molar excess of polymer maleimide isemployed, for example, a 1.5 to 15-fold molar excess, preferably a2-fold to 10 fold molar excess. Reaction times generally range fromabout 15 minutes to several hours, e.g., 8 or more hours, at roomtemperature. For sterically hindered sulfhydryl groups, requiredreaction times may be significantly longer. The stabilized maleimides ofthe invention are thiol-selective, and thiol-selective conjugation ispreferably conducted at pHs around 7.

[0263] Reactions with amino groups proceed at higher pHs, but arerelatively slow. Protein PEGylation reaction conditions vary dependingon the protein, the desired degree of PEGylation, and the particularpolymer maleimide reagent.

[0264] Separation

[0265] Optionally, conjugates produced by reacting a PEG maleimide orPEG amine of the invention with a biologically active agent are purifiedto obtain/isolate different PEGylated species. Alternatively, and morepreferably for lower molecular weight PEGs, e.g., having molecularweights less than about 20 kilodaltons, preferably less than or equal toabout 10 kilodaltons, a product mixture can be purified to obtain adistribution around a certain number of PEGs per protein molecule, whereapplicable. For example, a product mixture can be purified to obtain anaverage of anywhere from one to five PEGs per protein, typically anaverage of about 3 PEGs per protein. The strategy for purification ofthe final conjugate reaction mixture will depend upon a number offactors—the molecular weight of the polymer employed, the particularprotein, the desired dosing regimen, and the residual activity and invivo properties of the individual conjugate(s) species.

[0266] If desired, PEG conjugates having different molecular weights canbe isolated using gel filtration chromatography. While this approach canbe used to separate PEG conjugates having different molecular weights,this approach is generally ineffective for separating positional isomershaving different pegylation sites within a protein. For example, gelfiltration chromatography can be used to separate from each othermixtures of PEG 1-mers, 2-mers, 3-mers, etc., although each of therecovered PEG-mer compositions may contain PEGs attached to differentreactive amino groups (e.g., lysine residues) within the protein.

[0267] Gel filtration columns suitable for carrying out this type ofseparation include Superdex™ and Sephadex™ columns available fromAmersham Biosciences. Selection of a particular column will depend uponthe desired fractionation range desired. Elution is generally carriedout using a non-amine based buffer, such as phosphate, acetate, or thelike. The collected fractions may be analysed by a number of differentmethods, for example, (i) OD at 280 nm for protein content, (ii) BSAprotein analysis, (iii) iodine testing for PEG content (Sims G. E. C.,et al., Anal. Biochem, 107, 60-63, 1980), or alternatively, (iv) byrunning an SDS PAGE gel, followed by staining with barium iodide.

[0268] Separation of positional isomers is carried out by reverse phasechromatography using an RP-HPLC C18 column (Amersham Biosciences orVydac) or by ion exchange chromatography using an ion exchange column,e.g., a Sepharose™ ion exchange column available from AmershamBiosciences. Either approach can be used to separate PEG-biomoleculeisomers having the same molecular weight (positional isomers).

[0269] Depending upon the intended use for the resulting PEG-conjugates,following conjugation, and optionally additional separation steps, theconjugate mixture may be concentrated, sterile filtered, and stored atlow temperatures from about −20° C. to about −80° C. Alternatively, theconjugate may be lyophilized, either with or without residual buffer andstored as a lyophilized powder. In some instances, it is preferable toexchange a buffer used for conjugation, such as sodium acetate, for avolatile buffer such as ammonium carbonate or ammonium acetate, that canbe readily removed during lyophilization, so that the lyophilizedprotein conjugate powder formulation is absent residual buffer.Alternatively, a buffer exchange step may be used using a formulationbuffer, so that the lyophilized conjugate is in a form suitable forreconstitution into a formulation buffer and ultimately foradministration to a mammal.

[0270] Target Molecules and Surfaces

[0271] The stabilized polymer maleimides (amines) of the invention maybe attached, either covalently or non-covalently, to a number ofentities including films, chemical separation and purification surfaces,solid supports, metal/metal oxide surfaces such as gold, titanium,tantalum, niobium, aluminum, steel, and their oxides, silicon oxide,macromolecules, and small molecules. Additionally, the polymers of theinvention may also be used in biochemical sensors, bioelectronicswitches, and gates. The polymer maleimides (amines) of the inventionmay also be employed as carriers for peptide synthesis, for thepreparation of polymer-coated surfaces and polymer grafts, to preparepolymer-ligand conjugates for affinity partitioning, to preparecross-linked or non-cross-linked hydrogels, and to preparepolymer-cofactor adducts for bioreactors.

[0272] A biologically active agent for use in coupling to a polymer ofthe invention may be any one or more of the following. Suitable agentsmay be selected from, for example, hypnotics and sedatives, psychicenergizers, tranquilizers, respiratory drugs, anticonvulsants, musclerelaxants, antiparkinson agents (dopamine antagnonists), analgesics,anti-inflammatories, antianxiety drugs (anxiolytics), appetitesuppressants, antimigraine agents, muscle contractants, anti-infectives(antibiotics, antivirals, antifungals, vaccines) antiarthritics,antimalarials, antiemetics, anepileptics, bronchodilators, cytokines,growth factors, anti-cancer agents, antithrombotic agents,antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants,anti-asthma agents, hormonal agents including contraceptives,sympathomimetics, diuretics, lipid regulating agents, antiandrogenicagents, antiparasitics, anticoagulants, neoplastics, antineoplastics,hypoglycemics, nutritional agents and supplements, growth supplements,antienteritis agents, vaccines, antibodies, diagnostic agents, andcontrasting agents.

[0273] More particularly, the active agent may fall into one of a numberof structural classes, including but not limited to small molecules(preferably insoluble small molecules), peptides, polypeptides,proteins, antibodies, polysaccharides, steroids, nucleotides,oligonucleotides, polynucleotides, fats, electrolytes, and the like.Preferably, an active agent for coupling to a polymer maleimide of theinvention possesses a native amino or a sulfydryl group, oralternatively, is modified to contain at least one reactive amino orsulfhydryl group suitable for coupling to a polymer maleimide of theinvention.

[0274] Specific examples of active agents suitable for covalentattachment to a polymer of the invention include but are not limited toaspariginase, amdoxovir (DAPD), antide, becaplermin, calcitonins,cyanovirin, denileukin diftitox, erythropoietin (EPO), EPO agonists(e.g., peptides from about 10-40 amino acids in length and comprising aparticular core sequence as described in WO 96/40749), dornase alpha,erythropoiesis stimulating protein (NESP), coagulation factors such asFactor V, Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X,Factor XII, Factor XIII, von Willebrand factor; ceredase, cerezyme,alpha-glucosidase, collagen, cyclosporin, alpha defensins, betadefensins, exedin-4, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), fibrinogen,filgrastim, growth hormones human growth hormone (hGH), growth hormonereleasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenicproteins such as bone morphogenic protein-2, bone morphogenic protein-6,OP-1; acidic fibroblast growth factor, basic fibroblast growth factor,CD-40 ligand, heparin, human serum albumin, low molecular weight heparin(LMWH), interferons such as interferon alpha, interferon beta,interferon gamma, interferon omega, interferon tau, consensusinterferon; interleukins and interleukin receptors such as interleukin-1receptor, interleukin-2, interluekin-2 fusion proteins, interleukin-1receptor antagonist, interleukin-3, interleukin-4, interleukin-4receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13receptor, interleukin-17 receptor; lactoferrin and lactoferrinfragments, luteinizing hormone releasing hormone (LHRH), insulin,pro-insulin, insulin analogues (e.g., mono-acylated insulin as describedin U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin,somatostatin analogs including octreotide, vasopressin, folliclestimulating hormone (FSH), influenza vaccine, insulin-like growth factor(IGF), insulintropin, macrophage colony stimulating factor (M-CSF),plasminogen activators such as alteplase, urokinase, reteplase,streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growthfactor (NGF), osteoprotegerin, platelet-derived growth factor, tissuegrowth factors, transforming growth factor-1, vascular endothelialgrowth factor, leukemia inhibiting factor, keratinocyte growth factor(KGF), glial growth factor (GGF), T Cell receptors, CDmolecules/antigens, tumor necrosis factor (TNF), monocytechemoattractant protein-1, endothelial growth factors, parathyroidhormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1,thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9,thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosponates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

[0275] Additional agents suitable for covalent attachment to a polymerof the invention include but are not limited to amifostine, amiodarone,aminocaproic acid, aminohippurate sodium, aminoglutethimide,aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide,anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide,bleomycin, buserelin, busulfan, cabergoline, capecitabine, carboplatin,carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine,clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins,13-cis retinoic acid, all trans retinoic acid; dacarbazine,dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril,lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, metaraminol bitartrate,methotrexate, metoclopramide, mexiletine, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

[0276] Preferred peptides or proteins for coupling to a polymermaleimide of the invention include EPO, IFN-α, IFN-β, IFN-γ, consensusIFN, Factor VII, Factor VIII, Factor IX, IL-2, remicade (infliximab),Rituxan (rituximab), Enbrel (etanercept), Synagis (palivizumab), Reopro(abciximab), Herceptin (trastuzimab), tPA, Cerizyme (imiglucerase),Hepatitus-B vaccine, rDNAse, alpha-1 proteinase inhibitor, GCSF, GMCSF,hGH, insulin, FSH, and PTH.

[0277] The above exemplary biologically active agents are meant toencompass, where applicable, analogues, agonists, antagonists,inhibitors, isomers, and pharmaceutically acceptable salt forms thereof.In reference to peptides and proteins, the invention is intended toencompass synthetic, recombinant, native, glycosylated, andnon-glycosylated forms, as well as biologically active fragmentsthereof. The above biologically active proteins are additionally meantto encompass variants having one or more amino acids substituted (e.g.,cysteine), deleted, or the like, as long as the resulting variantprotein possesses at least a certain degree of activity of the parent(native) protein.

[0278] Pharmaceutical Compositions

[0279] The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

[0280] Exemplary excipients include, without limitation, those selectedfrom the group consisting of carbohydrates, inorganic salts,antimicrobial agents, antioxidants, surfactants, buffers, acids, bases,and combinations thereof.

[0281] A carbohydrate such as a sugar, a derivatized sugar such as analditol, aldonic acid, an esterified sugar, and/or a sugar polymer maybe present as an excipient. Specific carbohydrate excipients include,for example: monosaccharides, such as fructose, maltose, galactose,glucose, D-mannose, sorbose, and the like; disaccharides, such aslactose, sucrose, trehalose, cellobiose, and the like; polysaccharides,such as raffinose, melezitose, maltodextrins, dextrans, starches, andthe like; and alditols, such as mannitol, xylitol, maltitol, lactitol,xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and thelike.

[0282] The excipient can also include an inorganic salt or buffer suchas citric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

[0283] The preparation may also include an antimicrobial agent forpreventing or deterring microbial growth. Nonlimiting examples ofantimicrobial agents suitable for the present invention includebenzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate, thimersol, and combinations thereof.

[0284] An antioxidant can be present in the preparation as well.Antioxidants are used to prevent oxidation, thereby preventing thedeterioration of the conjugate or other components of the preparation.Suitable antioxidants for use in the present invention include, forexample, ascorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite,and combinations thereof.

[0285] A surfactant may be present as an excipient. Exemplarysurfactants include: polysorbates, such as “Tween 20” and “Tween 80,”and pluronics such as F68 and F88 (both of which are available fromBASF, Mount Olive, N.J.); sorbitan esters; lipids, such as phospholipidssuch as lecithin and other phosphatidylcholines,phosphatidylethanolamines (although preferably not in liposomal form),fatty acids and fatty esters; steroids, such as cholesterol; andchelating agents, such as EDTA, zinc and other such suitable cations.

[0286] Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

[0287] The pharmaceutical preparations encompass all types offormulations and in particular those that are suited for injection,e.g., powders that can be reconstituted as well as suspensions andsolutions. The amount of the conjugate (i.e., the conjugate formedbetween the active agent and the polymer described herein) in thecomposition will vary depending on a number of factors, but willoptimally be a therapeutically effective dose when the composition isstored in a unit dose container (e.g., a vial). In addition, thepharmaceutical preparation can be housed in a syringe. A therapeuticallyeffective dose can be determined experimentally by repeatedadministration of increasing amounts of the conjugate in order todetermine which amount produces a clinically desired endpoint.

[0288] The amount of any individual excipient in the composition willvary depending on the activity of the excipient and particular needs ofthe composition. Typically, the optimal amount of any individualexcipient is determined through routine experimentation, i.e., bypreparing compositions containing varying amounts of the excipient(ranging from low to high), examining the stability and otherparameters, and then determining the range at which optimal performanceis attained with no significant adverse effects.

[0289] Generally, however, the excipient will be present in thecomposition in an amount of about 1% to about 99% by weight, preferablyfrom about 5%-98% by weight, more preferably from about 15-95% by weightof the excipient, with concentrations less than 30% by weight mostpreferred.

[0290] These foregoing pharmaceutical excipients along with otherexcipients are described in “Remington: The Science & Practice ofPharmacy”, 19^(th) ed., Williams & Williams, (1995), the “Physician'sDesk Reference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998),and Kibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

[0291] The pharmaceutical preparations of the present invention aretypically, although not necessarily, administered via injection and aretherefore generally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

[0292] As previously described, the conjugates can be administeredinjected parenterally by intravenous injection, or less preferably byintramuscular or by subcutaneous injection. Suitable formulation typesfor parenteral administration include ready-for-injection solutions, drypowders for combination with a solvent prior to use, suspensions readyfor injection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

[0293] Methods of Administering

[0294] The invention also provides a method for administering aconjugate as provided herein to a patient suffering from a conditionthat is responsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

[0295] The unit dosage of any given conjugate (again, preferablyprovided as part of a pharmaceutical preparation) can be administered ina variety of dosing schedules depending on the judgment of theclinician, needs of the patient, and so forth. The specific dosingschedule will be known by those of ordinary skill in the art or can bedetermined experimentally using routine methods. Exemplary dosingschedules include, without limitation, administration five times a day,four times a day, three times a day, twice daily, once daily, threetimes weekly, twice weekly, once weekly, twice monthly, once monthly,and any combination thereof. Once the clinical endpoint has beenachieved, dosing of the composition is halted.

[0296] One advantage of administering the conjugates of the presentinvention is that individual water-soluble polymer portions can becleaved off. Such a result is advantageous when clearance from the bodyis potentially a problem because of the polymer size. Optimally,cleavage of each water-soluble polymer portion is facilitated throughthe use of physiologically cleavable and/or enzymatically degradablelinkages such as urethane, amide, carbonate or ester-containinglinkages. In this way, clearance of the conjugate (via cleavage ofindividual water-soluble polymer portions) can be modulated by selectingthe polymer molecular size and the type functional group that wouldprovide the desired clearance properties. One of ordinary skill in theart can determine the proper molecular size of the polymer as well asthe cleavable functional group. For example, one of ordinary skill inthe art, using routine experimentation, can determine a proper molecularsize and cleavable functional group by first preparing a variety ofpolymer derivatives with different polymer weights and cleavablefunctional groups, and then obtaining the clearance profile (e.g.,through periodic blood or urine sampling) by administering the polymerderivative to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

[0297] All articles, books, patents, patent publications and otherpublications referenced herein are incorporated by reference in theirentireties.

EXAMPLES

[0298] It is to be understood that while the invention has beendescribed in conjunction with certain preferred specific embodimentsthereof, the foregoing description as well as the examples that followare intended to illustrate and not limit the scope of the invention.Other aspects, advantages and modifications within the scope of theinvention will be apparent to those skilled in the art to which theinvention pertains.

[0299] ABBREVIATIONS.

[0300] DCM: dichloromethane

[0301] NMR: nuclear magnetic resonance

[0302] DI: deionized

[0303] r.t. room temperature

[0304] anh. Anhydrous

[0305] Da Daltons

[0306] GPC gel permeation chromatography

[0307] Materials and Methods.

[0308] All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated.

[0309] All PEG reagents referred to in the appended examples areavailable from Nektar, Huntsville, Ala. All ¹HNMR data was generated bya 300 or 400 MHz NMR spectrometer manufactured by Bruker.

Example 1 BRANCHED PEG2-AMIDOPENTAMETHYLENE-MALEIMIDE (40 KDA) (L1-AMPE)

[0310] Overview of Synthesis:

[0311] A. Branched PEG2(40 K)Amine

[0312] To a solution of branched PEG2(40,000)—N-hydroxysuccinimide ester(20 g, 0.00050 moles) (Nektar, Huntsville Ala.) in methylene chloride(250 ml), ethylenediamine (0.68 ml, 0.01017 moles) was added and thereaction mixture was stirred overnight at room temperature under argonatmosphere. Next the solvent was evaporated to dryness. The crudeproduct was dissolved in small amount of methylene chloride andprecipitated with isopropyl alcohol. The wet product was dried underreduced pressure. Yield 17.2 g.

[0313] NMR (d₆-DMSO): 2.65 ppm (t, —CH ₂—NH₂), 3.24 ppm (s, —OCH₃), 3.51ppm (s, PEG backbone).

[0314] B. Branched PEG2-amidopentamethylene-maleimide-40 kDa

[0315] To a solution of N-succinimidyl(ε-maleimidohexanoate) (0.1 g,0.000324 moles, Pierce Chemical Company), in methylene chloride (10 ml),was added over a period of 3 minutes a solution of branched PEG2 (40K)amine from Step A (12.2 g, 0.000305 moles) in methylene chloride (20ml). Next, 0.045 ml of triethylamine was added and the mixture wasstirred overnight at room temperature under an argon atmosphere. Thesolvent was then distilled and the crude product that remained wasdissolved in 30 ml of methylene chloride and then precipitated by theaddition of 450 ml of isopropyl alcohol at room temperature. The yieldwas 11.5 g.

[0316] Proton NMR analysis indicated main signals at: 3.24 ppm (s,—OCH₃), 3.51 ppm (s, PEG backbone), 7.01 ppm (s, CH═CH, maleimide),which are indicative of the correct product. On the basis of the NMR,the substitution was estimated to be approximately 89%. GPC analysisrevealed the main product to be 98.3% desired compound, with 1.7% dimer.The product also possessed required ultraviolet absorption.

Example 2 MPEG(5,000 DA)-BUTYLMALEIMIDE (L3-TEME)

[0317] Overview of Synthesis:

[0318] A. mPEG(5,000 Da)-butylamine

[0319] A solution of mPEG-5,000 Da (2.0 g, 0.0004 moles) (NOFCorporation) in toluene (30 ml) was azeotropically dried by distillingoff 15 ml toluene. 1.0M solution of potassium tert-butoxide intert-butanol (2.0 ml, 0.002 moles) and 1,4-dibromobutane (0.43 g, 0.002moles) were added and the mixture was stirred overnight at 75° C. underargon atmosphere. The mixture was filtered and the solvents weredistilled off under reduced pressure. The residue was dissolved indichloromethane (3 ml) and isopropyl alcohol (50 ml) was added. Theprecipitated product was filtered off and dried under reduced pressure.Next it was dissolved in concentrated ammonia (20 ml) and the resultingsolution was stirred 20 h at room temperature. The product was extractedwith dichloromethane. The extract was dried with anhydrous magnesiumsulfate and the solvent was distilled off under reduced pressure giving1.5 g of M-PEG(5,000)-butylamine.

[0320] NMR (D₂O): 1.53 ppm (m, —CH₂ —CH ₂—CH₂—NH₂) 2.75 ppm (t, —CH₂—NH₂), 3.27 ppm (s, —OCH₃), 3.53 ppm (s, PEG backbone).

[0321] B. mPEG(5000 Da)-butylmaleimide

[0322] mPEG(5,000)-butylamine (1.0 g, 0.0002 moles) from Step A wasdissolved in saturated aqueous NaHCO₃ (5 ml) and the mixture was cooledto ° C. N-methoxycarbonylmaleimide (0.25 g) was added with vigorousstirring. After stirring for 15 minutes, water (8 ml) was added and themixture was stirred an additional 65 minutes. NaCl (0.5 g) was added andthe pH was adjusted to 3.0 with 10% phosphoric acid. The product wasextracted with dichloromethane. The extract was dried with anhydrousMgSO₄ and the solvent was distilled off under reduced pressure giving0.9 g of white, solid product.

[0323] NMR (d₆-DMSO): 1.48 ppm (bm, —CH₂ —CH ₂—CH₂-Mal), 3.24 ppm (s,—OCH₃), 3.51 ppm (s, PEG backbone), 7.00 ppm (s, —CH═CH—).

[0324] The product had 82% substitution of the maleimidyl group on thePEG moiety.

Example 3 MPEG(5000 DA)-HEXYLMALEIMIDE (L3-HEXA)

[0325]

[0326] This synthesis is essentially equivalent to that described inExample 2 above, with the exception that the dibromo-reagent utilizedpossesses two additional methylenes, i.e., Br—(CH₂)₆—Br.

[0327] A. mPEG(20,000 Da)-hexylamine

[0328] A solution of mPEG-5,000 Da (2.0 g, 0.0004 moles) (NOFCorporation) in toluene (30 ml) was azeotropically dried by distillingoff 15 ml toluene. 1.0M solution of potassium tert-butoxide intert-butanol (2.0 ml, 0.002 moles) and 1,6-dibromohexane (0.49 g, 0.002moles) were added and the mixture was stirred overnight at 80° C. underargon atmosphere. The mixture was filtered and the solvents weredistilled off under reduced pressure. The residue was dissolved indichloromethane (3 ml) and isopropyl alcohol (50 ml) was added. Theprecipitated product was filtered off and dried under reduced pressure.Next it was dissolved in concentrated ammonia (20 ml) and the resultingsolution was stirred 20 h at room temperature. The product was extractedwith dichloromethane. The extract was dried with anhydrous magnesiumsulfate and the solvent was distilled off under reduced pressure giving1.6 g of M-PEG(5,000)-hexylamine.

[0329] NMR (D₂O): 1.28 ppm (m, —CH₂ —CH ₂—CH₂—CH₂—NH₂), 1.47 ppm (m, —CH₂—CH ² —CH₂—CH ₂—CH₂—NH₂), 2.71 ppm (t, —CH ₂—NH₂), 3.27 ppm (s, —OCH₃),3.53 ppm (s, PEG backbone).

[0330] B. mPEG(5000 Da)-hexylmaleimide

[0331] mPEG(5,000 Da)-hexylamine (1.0 g, 0.0002 moles) from Step A wasdissolved in saturated aqueous NaHCO₃ (5 ml) and the mixture was cooledto ° C. N-methoxycarbonylmaleimide (0.25 g) was added with vigorousstirring. After stirring for 15 minutes, water (8 ml) was added and themixture was stirred an additional 65 minutes. NaCl (0.5 g) was added andthe pH was adjusted to 3.0 with 10% phosphoric acid. The product wasextracted with dichloromethane. The extract was dried with anhydrousMgSO₄ and the solvent was distilled off under reduced pressure giving0.9 g of white, solid product.

[0332] NMR (d₆-DMSO): 1.24 ppm (bm, —CH₂ —CH ₂—CH₂—CH₂-Mal), 1.45 ppm(bm, —CH₂ —CH₂—CH₂—CH ₂—CH₂-Mal), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEGbackbone), 7.01 ppm (s, —CH═CH—).

[0333] The product had 80% substitution of the maleimidyl group on thePEG moiety.

Example 4 MPEG (5 K DA)-PROPYLMALEIMIDE (L3-TME)

[0334] Overview of Synthesis:

[0335] A. MPEG (5 K Da)-Propylamine

[0336] To a solution of 4,7,10-trioxa-1,13-tridecanediamine (4.2 g) inanh. acetonitrile (100 ml) was added mPEG-benzotriazolylcarbonate (5 g)(Shearwater Corp.) in anh. acetonitrile (60 ml) during 20 min and themixture was stirred overnight at room temperature under argonatmosphere. Next the solvent was distilled off. The product wasdissolved in 100 ml DI H₂O. NaCl (5 g) was added and the pH was adjustedto 3.0 with 10% H₃PO₄. The product was extracted with CH₂Cl₂. Theextract was washed with 50 ml 2% KOH solution, then it was dried (MgSO₄)and the solvent was distilled off. Next the product was dissolved in 10ml CH₂Cl₂ and reverse precipitated with 200 ml isopropyl alcohol at 0-5°C. Yield after drying 4.2 g.

[0337] NMR: Desired product, substitution 85.0%, GPC (buffer, 25° C.)substitution 97.02%.

[0338] B. MPEG (5K Da)-PA-Maleimide

[0339] mPEG (5K Da)-propylamine (4.0 g in 20 ml deionized water, pH of8.93) from Step A was cooled to 0-5° C. on an ice bath and a solution ofN-methoxycarbonylmaleimide (0.5 g in 3.5 ml of anh. acetonitrile) wasadded and the mixture was stirred 15 min at 0-5° C. The ice bath wasremoved and DI H₂O (16 ml) was added and the mixture was stirred 45 minat room temperature. NaCl (2 g) was added and the pH of the mixture wasadjusted to 3.0 with 10% H₃PO₄. The product was extracted with CH₂Cl₂.The extract was dried with MgSO₄ and the solvent was distilled off. Thecrude product was dissolved in CH₂Cl₂ (10 ml) and precipitated withisopropyl alcohol (200 ml) at 0-5° C. Yield 3.7 g.

[0340] NMR: Confirmed synthesis of desired product; substitution 83.5%.

Example 5 MPEG(5 K DA)-AMIDOCYCLOHEXYLMETHYL-MALEIMIDE (L1-MCH)

[0341] Overview of Synthesis:

[0342] To a solution of 4-(maleimidomethyl)-1-cyclohexanecarboxylicacid, NHS ester (0.100 g) (Pierce Chemical Company) in CH₂Cl₂ (10 ml)was added a solution of mPEG (5K Da)-amine (1.5 g) (Shearwater Corp.) inCH₂Cl₂ (20 ml). TEA (0.042 ml) was added and the mixture was stirredovernight at room temperature under argon atmosphere. The solvent wasdistilled off. The crude product was dissolved in 2 ml CH₂Cl₂ andprecipitated with isopropyl alcohol (60 ml) at 0-5° C. Yield 1.35 g.

[0343] NMR: Desired compound, substitution 79.7%. GPC (nitrate buffer,25° C.): dimer: 3.98%; Main compound: 96.02%.

Example 6 CONJUGATE OF MPEG (5 K DA)-PROPYLMALEIMIDE (L3-TME)

[0344] To illustrate reaction of a reactive polymer of the inventionwith a molecule bearing a thiol group, to a solution of MPEG-PA-MAL fromExample 4 (1.0 g) in phosphate buffer was added 25 μl of2-mercaptoethanol. The mixture was stirred overnight at room temperatureunder argon atmosphere. The product was extracted with CH₂Cl₂ (3×20 ml).The extract was dried (MgSO4) and the solvent was distilled off. Thecrude product was dissolved in 2 ml CH₂Cl₂ and precipitated with 40 mlisopropyl alcohol at 0-5° C. Yield 0.78 g

[0345] NMR: Formation of the desired product was confirmed by NMR;substitution: 64.9%

Example 7 HYDROLYSIS RATE STUDY OF REACTIVE POLYMERS

[0346] Using HPLC analysis, the rate of hydrolytic degradation of themaleimide ring of several exemplary maleimide-terminated MPEG polymers(average molecular weight 5000 Da) was explored.

[0347] The following linkages between the maleimide and the PEG polymersegment were evaluated: amidoethylene (L1-AMDE), amidopentamethylene(L1-AMPE), amidocyclohexylmethyl (L1-MCH), oxybutyl (L3-TEME), oxyhexyl(L3-HEXA), oxyethyl (L3-ET), and oxypropyl (L3-TME). Complete structuresare provided below for ease of reference. TABLE 2 Hydrolysis Rates ofmPEG (5 k-Da) Maleimides (5 mg/ml) in 50 mM Phosphate Buffer (pH˜7.5) asMeasured by UV Absorption at 297 nm

mPEG-AMDE-MAL

mPEG-AMPE-MAL

mPEG-MCH-MAL

mPEG-TEME-MAL

mPEG-HEXA-MAL

mPEG-ET-MAL

mPEG-TME-MAL Polymer Structure (Table 1) Half-life (hrs) RelativeHydrolysis Rate mPEG-AMDE-MAL L1-AMDE 8.8 3.66 mPEG-AMPE-MAL L1-AMPE19.4 1.66 mPEG-MCH-MAL L1-MCH 16.3 1.98 mPEG-TEME-MAL L3-TEME 19.6 1.65mPEG-HEXA-MAL L3-HEXA 32.3 1.00 mPEG-ET-MAL L3-ET 8.1 4.01 mPEG-TME-MALL3-TME 11.5 2.82

[0348] As can be shown by the data in Table 2 above, the hydrolysisrates of these illustrative polymeric maleimides to form theirrespective maleamic acids varies with changes in the structure of thehydrocarbon portion adjacent to the maleimide ring. The data in columnthree demonstrates rates of hydrolysis relative to thehexamethylene-maleimide polymer. As can be seen, for the polymersexamined, the L3-HEXA polymer was the most stable, that is to say, hadthe slowest hydrolysis rate, and thus, the longest half life. The dataabove indicates that an increase in the length of the hydrocarbon chainseparating the polymer and the maleimide increases the half-life of themaleimide-terminated polymer itself.

Example 8 HYDROLYSIS RATE STUDY OF POLYMER CONJUGATES

[0349] The hydrolysis rates of representative protein and small moleculemodel conjugates were investigated to examine the correlation betweenthe ring opening tendencies of the polymer-terminated maleimidesthemselves versus their conjugates.

[0350] Since large biomolecular components such as proteins have adramatic effect on the retention of conjugated molecules on commonliquid chromatography columns, it is generally more difficult to measurekinetics of maleimide conjugates than it is for the polymers themselves.In this analysis, the open acid form of the maleamic acid was notdistinctly separable from the unopened or closed ring form. However, acombination analysis based upon size exclusion chromatography (HPLC-SE)and analytical protein electrophoresis (SDS-PAGE) was successfullyemployed to estimate the ring opening characteristics of polymericmaleimide protein conjugates, as well as conjugates prepared using modelnon-protein compounds.

[0351] In this study, two PEG-globular protein conjugates representedgenerally below were studied to examine their ring openingcharacteristics.

[0352] The top structure is a PEG-maleimide conjugate of Glob Protein 2,where Glob Protein 2 is a protein having a molecular weight ofapproximately 48 kDa. Glob Protein 2 was conjugated to a PEG maleimidederived from a PEG propionic acid, MW 30 kDa, which further included amedium-length linker interposed between the propionic acid derivedportion of the polymer and the maleimide terminus. The linker in the topstructure is —C(O)—NH(CH₂)₂—NH—C(O)—CH₂CH₂—.

[0353] The bottom structure is a PEG-maleimide conjugate of Glob Protein1, where the protein possesses a molecular weight of about 11 kDa. Theconjugate was prepared using a linkerless maleimide (mPEG-ET-MAL) havinga molecular weight of about 20 kDa. The corresponding PEG maleimidestructure is L3-ET, discussed in Example 7.

[0354] The bottom structure (Glob Protein 2) is completely ring openedafter 24 hours at pH 8.5 at room temperature, thus indicating theinstability of this type of maleimidyl terminated polymer absent astabilizing linker separating the polymer and the maleimide ring.Relative to the linkerless form, however, the linker in the topstructure (Glob Protein 1) retards the ring opening, since the ringstructure in the top conjugate is not completely ring-opened until 17hours, at pH 9, upon heating to 50° C. for 17 hours.

[0355] Similarly, hydrolysis rates of the stabilized polymer maleimidesof the invention conjugated to a model compound, 2-mercaptoethanol, weredetermined to assess the tendency of the conjugates towardsring-opening. The study revealed that the present stabilized polymermaleimides are superior to those used to form conjugates of Glob Protein1 and Glob Protein 2. That it to say, in both free and in conjugatedform, the polymer maleimides of the invention exhibited superiorstability and resistance against ring opening in comparison tolinkerless PEG maleimide and, for example, the PEG-maleimide above,shown attached to Glob Protein 2.

[0356] Hydrolysis rate studies of conjugates of 8-TRI, 8-PEN, and 8-MCH(structures provided below in Table 3) were conducted as described abovefor the unconjugated maleimides. The half-lives shown were calculatedfrom data taken at two different pH values. Similar to the unconjugatedmaleimides, the data indicate a slowing in reaction rate as the pHsdrifted lower with increased ring opening. The linkage with the shortesthydrocarbon chain adjacent to the succinimide ring (i.e., 8-TRI) was thefastest to open in comparison to the other conjugates studied. The dataindicate that longer/larger hydrocarbon chains provide superiorresistance to hydrolysis-induced ring opening. TABLE 3 HydrolysisHalf-lives of mPEG (5 k-Da) Maleimide Conjugates

Experimentally Linker, Determined Half-lives D pH 9.06 pH 8.11 8-TRI; D= trimethylene 31.4 hours 17.6 days 8-PEN; D = pentamethylene — 28.5days

43.3 hours —

Example 9 SYNTHESIS OF 1-(N-MALEIMIDOMETHYL)-4-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDOMETHYL)CYCLOHEXANE (MIXTURE OF CIS AND TRANS ISOMERS)

[0357] Step 1.

[0358] 9A. Preparation of 1-aminomethyl-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (20.0 g, 4.0 mmol, Nektar Therapeutics)in acetonitrile (200 mL) was added dropwise to a solution of1,4-cyclohexane(bismethylamine) (11.34 g, 79.7 mmol) in acetonitrile(200 mL) containing triethylamine (20 mL). This mixture was stirred atroom temperature for 3 days. The solvent was removed in vacuo leaving awhite solid. The solid was stirred with ether (100 mL), collected byfiltration and dried to yield 20.23 g of a crude product. This crudemixture was taken up in CH₂Cl₂ (30 mL) and precipitated with IPA (500mL)/ether (250 mL). The solid was collected by filtration and driedunder vacuum (16.3 g).

[0359]¹H NMR (dmso-d₆) δ 7.76 (1H, d, NHC═O), 3.51 (br s, O—CH₂CH₂—, PEGbackbone), 2.98 and 2.88 (2H, t, CH ₂—NH—C═O), 2.43 and 2.36 (2H, d, CH₂—NH₂), 2.30 (2H, CH₂C═O), 1.78-1.68 (1H, m, ring CH), 1.45-1.21 (6H, m,ring methylene protons), 0.90-0.73 (1H, m, ring CH).

[0360] Step 2.

[0361] 9.B. Preparation of 1-(N-maleimidomethyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): A solution of 1-aminomethyl-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)(3.68 g, 0.74 mmol) in NaHCO₃ (sat'd, 19 mL) was cooled in anice/salt/water bath. To this was added N-methoxycarbonylmaleimide (116mg, 0.82 mmol). This mixture was stirred in the ice bath for 15 minutesand H₂O (29 mL) was added. After stirring in the ice bath for 1 h, thereaction mixture was removed from the bath and stirred at RT for 3 h.The reaction mixture was diluted with brine (30 mL). The pH was adjustedto 3 with 10% phosphoric acid and extracted with CH₂Cl₂ (3×50 mL). Thecombined organic extracts were dried (Na₂SO₄) and concentrated in vacuo.The residue was taken up in CH₂Cl₂ (10 mL) and precipitated with IPA (60mL)/ether (100 mL). The product was collected by filtration and driedunder vacuum overnight (3.14 g).

[0362]¹H NMR (dmso-d₆) δ 7.79 (1H, d, NHC═O), 7.01 (2H, s, CH═CH), 3.51(br s, O—CH₂CH₂—, PEG backbone), 2.32 (2H, CH₂C═O), 1.78-1.45 (2H, m,ring methylene), 1.45-1.21 (5H, m, ring methylene protons and CH),0.90-0.73 (1H, m, ring protons).

Example 10 SYNTHESIS OF TRANS-4-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDOMETHYL)-N-CYCLOHEXYLMALEIMIDE.

[0363] Step 1.

[0364] 10.A. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexyl-t-BOC amine: To a solution oftrans-4-aminomethylcyclohexyl-t-BOC-amine (1.0 g, 4.67 mmol, AlbanyMolecular) and N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (22.9 g, 4.20 mmol, Nektar Therapeutics)in Acetonitrile (200 mL) was added triethylamine (1.2 mL, 8.6 mmol)under Argon. This mixture was stirred at room temperature under an argonatmosphere for 24 h. NMR did not show any remaining protons from the SPAgroup. The solvent was removed in vacuo to give a white residue whichwas taken up in CH₂Cl₂ (60 mL) and precipitated with IPA (500 mL)/ether(1 L). The solid was collected by filtration and dried under vacuum toyield the product as a white solid (22.2 g).

[0365]¹H NMR (dmso-d₆) δ 7.80 (1H, s, CH—NH), 6.70 (1H, d, CH—NH), 3.55(br s, O—CH₂CH₂—, PEG backbone), 3.28 (3H, s, CH₃), 3.15 (1H, br s, CH),2.90 (2H, t, CH—CH ₂—NH), 2.33 (3H, t, CH₂—C═O), 1.74-1.65 (4H, M, ringprotons), 1.37 (9H, s, C(CH₃)₃), 1.25 (1H, br s, CH), 1.14-0.83 (4H, m,ring protons).

[0366] Step 2.

[0367] 10.B. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexylamine, trifluoroacetate. To asolution of trans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexyl-t-BOC amine (1.20 g, 0.24 mmol) inanhydrous CH₂Cl₂ (5.0 mL) was added trifluoroacetic acid (2.5 mL, 32.5mmol). This mixture was stirred at 25° C. for 18 h. The solvent wasremoved in vacuo leaving an oily residue which was dried overnight undervacuum. The residue was stirred with anhydrous ether (20 mL). Theproduct was collected by filtration followed by drying (0.99 g).

[0368]¹H NMR (dmso-d₆) δ 8.13 (1H, br s, NH), 7.80 (3H, d, NH₃), 3.51(br s, O—CH₂CH₂—, PEG backbone and CH), 3.24 (3H, s, CH₃), 2.97 (1H, brs, CH), 2.27 (2H, t, CH₂C═O), 1.95-1.35 (2H, m, cyclohexane protons),1.34-1.25 (2H, m, cyclohexane protons), 1.40-1.25 (2H, m, cyclohexaneprotons), 1.25-1.13 (2H, m, cyclohexane protons).

[0369] Step 3.

[0370] 10.B. Preparation of trans-4-(Methoxypoly(ethyleneglycol)propionamidomethyl)-N-cyclohexylmaleimide.trans-4-(Methoxypoly(ethylene glycol)propionamidomethyl)cyclohexylamine,trifluoroacetate (3.0 g, 0.60 mmol) was taken up in NaHCO₃ (aq, sat'd,16 mL) and cooled to 2° C. in an ice/salt bath. To this was addedN-methoxycarbonyl maleimide (100 mg, 0.70 mmol). After stirring at 2° C.for 15 minutes, H₂O (24 mL) was added to the reaction mixture andstirring was continued for 4 h. Brine (50 mL) was added followed by pHadjustment to 3 using 10% Phosphoric acid. This mixture was extractedwith CH₂Cl₂ (3×50 mL). The combined organic extracts were dried(Na₂SO₄), concentrated in vacuo, and dried under vacuum. ¹H NMR showedthe product to be ca. 50% maleimide/50% ring-opened material, fromincomplete ring closure.

[0371]¹H NMR (dmso-d₆) δ 7.80 (1H, d, NH), 6.96 (2H, s, maleimideCH═CH), 3.51 (474, br s, PEG backbone and CH), 3.24 (3H, s, CH₃), 2.89(2H, t, CH₂), 2.30 (2H, t, CH₂C═O), 1.85-1.73 (2H, m, cyclohexaneprotons), 1.73-1.63 (2H, m, cyclohexane protons), 1.32 (1H, br s, CH),1.15-1.05 (2H, m, cyclohexane protons), 1.00-0.85 (2H, m, cyclohexaneprotons).

Example 11 SYNTHESIS OF TRANS-4-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDO)-N-CYCLOHEXYLMALEIMIDE.

[0372] Step 1.

[0373] 11.A. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamido)cyclohexyl-t-BOC amine: To a solution ofmono-t-BOC-trans-1,4-diaminocyclohexane (1.0 g, 4.38 mmol, AlbanyMolecular) and N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (21.5 g, 4.30 mmol, Nektar Therapeutics)in acetonitrile (200 mL) was added triethylamine (1.2 mL, 8.6 mmol)under Argon. This mixture was stirred at room temperature under an argonatmosphere for 24 h. ¹H NMR did not show any remaining protons from theN-hydroxysuccinimidyl ester group. The solvent was removed in vacuo togive a white residue which was stirred with ether (50 mL) for 30minutes. The solid was collected by filtration and dried under vacuum toyield the product as a white solid (1.95 g).

[0374]¹H NMR (dmso-d₆) δ 7.70 (1H, d, CH—NH), 6.69 (1H, d, CH—NH), 3.51(br s, O—CH₂CH₂—, PEG backbone and CH), 3.24 (3H, s, CH₃), 3.15 (1H, brs, CH), 2.26 (3H, t, CH₂—C═O), 1.74 (4H, br d, ring protons), 1.37 (9H,s, C(CH₃)₃), 1.17-1.07 (4H, m, ring protons).

[0375] Step 2.

[0376] 11.B. Preparation of trans-4-(methoxypoly(ethyleneglycol)propionamido)cyclohexylamine, trifluoroacetate. To a solution oftrans-4-(methoxypoly(ethylene glycol)propionamido)cyclohexyl-t-BOC amine(12.0 g, 2.4 mmol) in anhydrous CH₂Cl₂ (55 mL) was added trifluoroaceticacid (25 mL, 325 mmol). This mixture was stirred at 25° C. for 18 h. Thesolvent was removed in vacuo leaving an oily residue which was driedovernight under vacuum. The residue was taken up in CH₂Cl₂ (30 mL) andprecipitated with IPA (750 mL)/ether (500 mL). The product was collectedby filtration followed by drying to give the product as a white solid(10.2 g).

[0377]¹H NMR (dmso-d₆) δ 8.13 (1H, br s, NH), 7.80 (3H, d, NH₃), 3.51(br s, O—CH₂CH₂—, PEG backbone and CH), 3.24 (3H, s, CH₃), 2.97 (1H, brs, CH), 2.27 (2H, t, CH₂C═O), 1.95-1.35 (2H, m, cyclohexane protons),1.34-1.25 (2H, m, cyclohexane protons), 1.40-1.25 (2H, m, cyclohexaneprotons), 1.25-1.13 (2H, m, cyclohexane protons).

[0378] Step 3.

[0379] 11.C. Preparation of trans-4-(Methoxypoly(ethyleneglycol)propionamido)-N-cyclohexylmaleimide.trans-4-(Methoxypoly(ethylene glycol)propionamido)cyclohexylamine,trifluoroacetate (3.0 g, 0.60 mmol) was taken up in NaHCO₃ (aq, sat'd,16 mL) and cooled to 2° C. in an ice/salt bath. To this was addedN-methoxycarbonyl maleimide (100 mg, 0.70 mmol). After stirring at 2° C.for 15 minutes, H₂O (24 mL) was added to the reaction mixture andstirring was continued for 5 h. Brine (20 mL) was added followed by pHadjustment to 3 using 10% Phosphoric acid. This mixture was extractedwith CH₂Cl₂ (3×50 mL). The combined organic extracts were dried(Na₂SO₄), concentrated in vacuo, and dried under vacuum to give theproduct as a white solid (2.75 g). ¹H NMR showed the product to be 26%maleimide/74% opened material, from incomplete ring closure.

[0380]¹H NMR (dmso-d₆) δ 7.77 (1H, d, NH), 6.96 (2H, s, maleimideCH═CH), 3.51 (br s, O—CH₂CH₂—, PEG backbone), 3.24 (3H, s, CH₃), 2.28(2H, t, CH₂C═O), 2.06-1.92 (1H, m, cyclohexane proton), 1.88-1.73 (3H,m, cyclohexane protons), 1.59-1.65 (1H, m, cyclohexane proton),1.28-1.13 (3H, m, cyclohexane protons).

Example 12 SYNTHESIS OF 1-N-MALEIMIDOMETHYL-3-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDOMETHYL)CYCLOHEXANE (MIXTURE OF CIS AND TRANS ISOMERS)

[0381] Step 1.

[0382] 12.A. Preparation of 1-aminomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): N-hydroxysuccimidyl ester of methoxypoly(ethyleneglycol)propionic acid, MW 5,000 (10.0 g, 2.0 mmol, Nektar Therapeutics)in acetonitrile (100 mL) was added dropwise to a solution of1,3-cyclohexane(bismethylamine) (6.0 mL, 39.9 mmol, Albany Molecular) inacetonitrile (100 mL) containing triethylamine (10 mL). This mixture wasstirred at room temperature for 3 days. The insoluble solids werefiltered and the filtrate was concentrated in vacuo leaving a whitesolid. The crude mixture was taken up in CH₂Cl₂ (50 mL) and precipitatedwith IPA (375 mL)/ether (300 mL). The solid was filtered to give a whitesolid which was dried under vacuum (9.5 g). ¹H NMR showed some remaining1,3-cyclohexane(bismethylamine) which was removed by dissolving thesolid in CH₂Cl₂ and washing through Amberlyst 15 (15 g). The solvent wasremoved to give 1-methylamino-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (8.62 g).

[0383]¹H NMR (dmso-d₆) δ 7.78 (1H, d, NHC═O), 3.51 (br s, PEG backbone),3.24 (3H, s, OCH₃), 2.95 and 2.89 (2H, m, CH ₂—NH—C═O), 2.49 and 2.37(2H, d, CH ₂—NH₂), 2.30 (2H, CH₂C═O), 1.75-1.65 (4H, m, ring protons),1.63-1.21 (2H, m, ring protons), 1.21-1.03 (2H, m, ring protons),0.80-0.70 (1H, m, ring proton), 0.50-0.30 (1H, m, ring proton).

[0384] Step 2.

[0385] 12.B. Preparation of 1-N-maleimidomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): A solution of 1-aminomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl) cyclohexane (mixture of cis and transisomers) (4.0 g, 0.8 mmol) in NaHCO₃ (sat'd, 20 mL) was cooled in anice/salt/water bath. To this was added N-methoxycarbonylmaleimide (126mg, 0.89 mmol). This mixture was stirred in the ice bath for 15 minutesand H₂O (32 mL) was added. After stirring in the ice bath for 1 h, thereaction mixture was removed from the bath and stirred at RT for 3 h.The reaction mixture was diluted with brine (30 mL). The pH was adjustedto 3 with 10% phosphoric acid and extracted with CH₂Cl₂ (3×50 mL). Thecombined organic extracts were dried (Na₂SO₄) and concentrated in vacuo.The residue was dried under vacuum to give the product as a white solid.

[0386]¹H NMR (dmso-d₆) δ 7.79 (1H, d, NHC═O), 7.01 (2H, s, CH═CH), 3.51(br s, PEG backbone and CHCH ₂), 3.24 (3H, s, OCH₃) 3.10-2.63 (2H, m,CHCH ₂), 2.31 (2H, dd CH₂C═O), 1.65-1.1.10 (8H, m, ring protons),0.83-0.48 (2H, m, ring protons).

Example 13 Hydrolysis Rate Study of Exemplary Polymer Maleimides

[0387] Hydrolysis studies were conducted on several exemplary stabilizedPEG-maleimides as described above in Example 7. The structures of theparticular PEG-maleimides and their corresponding half-lives areprovided in Table 4 below. TABLE 4 Stability of Selected mPEG MaleimidesSTRUCTURE HALF-LIFE, IN HOURS

8.1

11.5

20.6

64.6

Example 14 CONJUGATION OF A STABILIZED MPEG-MALEIMIDE,1-(N-MALEIMIDOMETHYL)-4-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDOMETHYL)CYCLOHEXANE, TO THE MODEL COMPOUND,2-MERCAPTOETRANOL

[0388]

[0389] Preparation of1-(3-(2-hydroxyethanemercapto)-N-succinimidylmethyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)To a solution of 1-(N-maleimidomethyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)(500 mg, 0.1 mmol) in CH₃CN (10 mL) was added 2-mercaptoethanol (15 μL,0.21 mmol). This mixture was allowed to stir at room temperature for 18hours. ¹H NMR showed remaining maleimide starting material. Additional2-mercaptoethanol (15 μL, 0.21 mmol) was added and the mixture wasstirred for another 24 hours. ¹H NMR showed that no remaining maleimidewas present. The solvent was removed in vacuo and dried under vacuum.The solid was taken up in CH₂Cl₂ (2 mL) and precipitated with IPA (50mL). The solid was collected by filtration and dried to give the productas a white solid (403 mg).

[0390]¹H NMR (dmso-d₆) δ 7.66 (1H, br s, NH), 4.83 (1H, t, OH), 4.01(1H, dd, CH—S), 3.51 (br s, PEG backbone), 3.05-2.61 (4H, m, 2×CHCH ₂),2.30 (2H, t, CH₂C═O), 1.75-0.75 (10H, m, ring protons).

Example 15 CONJUGATION OF A STABILIZED MPEG-MALEIMIDE,TRANS-4-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDOMETHYL)-N-CYCLOHEXYLMALEIMIDE, TO THE MODEL COMPOUND,2-MERCAPTOETHANOL

[0391]

[0392] Preparation oftrans-1-(3-(2-hydroxyethanemercapto)-N-succinimidyl)-4-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane: To a solution oftrans-4-(methoxypoly(ethyleneglycol)propionamidomethyl)-N-cyclohexylmaleimide (400 mg, 0.0.08 mmol)in CH₃CN (10 mL) was added 2-mercaptoethanol (15 μL, 0.21 mmol). Thismixture was allowed to stir at room temperature for 18 hours. Thesolvent was removed in vacuo and dried under vacuum. The residue wasstirred with ether (2×20 mL) and the solid collected by filtration togive the product as a white solid (310 mg).

[0393]¹H NMR (dmso-d₆) δ 7.81 (1H, br s, NH), 4.87 (1H, t, OH), 3.85(1H, dd, CH—SEtOH), 3.51 (br s, PEG backbone, CH and SCH₂CH ₂OH), 3.24(3H, s, CH₃), 2.90 (2H, br s, CH₂NH), 2.79 (2H, t, SCH ₂CH₂), 2.30 (2H,t, CH₂C═O), 2.08-1.55 (5H, m, ring protons), 1.25-0.94 (4H, m, ringprotons).

Example 16 CONJUGATION OF A STABILIZED MPEG-MALEIMIDE,TRANS-4-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDO)-N-CYCLOHEXYLMALEIMIDE, TO THE MODEL COMPOUND,2-MERCAPTOETHANOL

[0394]

[0395] Preparation oftrans-1-(3-(2-hydroxyethanemercapto)-N-succinimidyl)-4-(methoxypoly(ethyleneglycol)propionamido)cyclohexane: To a solution oftrans-4-(methoxypoly(ethylene glycol)propionamido)-N-cyclohexylmaleimide(400 mg, 0.0.08 mmol) in CH₃CN (10 mL) was added 2-mercaptoethanol (15μL, 0.21 mmol). This mixture was allowed to stir at room temperature for18 hours. The solvent was removed in vacuo and dried under vacuum togive the product as a white solid (240 mg).

[0396]¹H NMR (dmso-d₆) δ 7.75 (1H, br s, NH), 4.83 (1H, t, OH), 4.01(1H, dd, CH—S), 3.51 (br s, PEG backbone and 2×CH), 2.75 (2H, m, S—CH₂),2.33 (2H, t, CH₂C═O), 2.02-1.65 (4H, m, ring protons), 1.55-0.95 (4H, m,ring protons).

Example 17 CONJUGATION OF A STABILIZED MPEG-MALEIMIDE,1-N-MALEIMIDOMETHYL-3-(METHOXYPOLY(ETHYLENEGLYCOL)PROPIONAMIDOMETHYL)CYCLOHEXANE, TO THE MODEL COMPOUND,2-MERCAPTOETHANOL

[0397]

[0398] Preparation of1-(3-(2-hydroxyethanemercapto)-N-succinimidylmethyl)-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and transisomers): To a solution of 1-N-maleimidomethyl-3-(methoxypoly(ethyleneglycol)propionamidomethyl)cyclohexane (mixture of cis and trans isomers)(450 mg, 0.0.09 mmol) in CH₃CN (10 mL) was added 2-mercaptoethanol (15μL, 0.21 mmol). This mixture was allowed to stir at room temperature for18 hours. The solvent was removed in vacuo and dried under vacuum. Theresidue was stirred with ether (20 mL) and the solid collected byfiltration to give the product as a white solid (230 mg).

[0399]¹H NMR (dmso-d₆) δ 7.81 (1H, br s, NH), 4.89 (1H, t, OH), 4.03(1H, dd, CH—S), 3.51 (PEG backbone and S—CH ₂CH ₂), 3.24 (3H, s, CH₃),3.05-2.60 (4H, m, 2×CHCH ₂), 2.30 (2H, t, CH ₂C═O), 1.80-1.05 (8H, m,ring protons), 0.80-0.51 (2H, m, ring protons).

[0400] Many modifications and other embodiments of the invention willcome to mind to one skilled in the art to which this invention pertainshaving the benefit of the teachings presented in the foregoingdescription. Therefore, it is to be understood that the invention is notto be limited to the specific embodiments disclosed and thatmodifications and other embodiments are intended to be included withinthe scope of the appended claims. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

What is claimed is:
 1. A water-soluble polymer having the structure:

wherein: POLY is a water-soluble polymer segment, b is 0 or 1, X is ahydrolytically stable linker comprising at least 3 contiguous saturatedcarbon atoms, and said polymer is absent aromatic groups and esterlinkages.
 2. The polymer of claim 1, wherein X is a saturated acyclic,cyclic or alicyclic hydrocarbon chain having a total of about 3 to about20 carbon atoms.
 3. The polymer of claim 2, wherein X is a saturatedacyclic, cyclic, or alicyclic hydrocarbon chain having a total number ofcarbon atoms selected from the group consisting of 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and
 20. 4. The polymer of claim3, wherein X is a saturated acyclic, cyclic, or alicyclic hydrocarbonchain having a total number of carbon atoms selected from the groupconsisting of: from about 3 to about 20, from about 4 to about 12, fromabout 4 to about 10, and from about 5 to about 8 atoms.
 5. The polymerof any one of claims 2 to 4, wherein X is a linear saturated acyclichydrocarbon chain.
 6. The polymer of claim 5, wherein X is a branchedsaturated acyclic hydrocarbon chain.
 7. The polymer of claim 6, whereinX is branched at the carbon α to the maleimidyl group.
 8. The polymer ofclaim 6, wherein X is branched at the carbon β to the maleimidyl group.9. The polymer of claim 6, wherein X is branched at the carbon γ to themaleimidyl group.
 10. The polymer of claim 5, having the structure:

wherein: y is an integer from 1 to about 20; R¹, in each occurrence, isindependently H or an organic radical that is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, andR², in each occurrence, is independently H or an organic radical that isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 11. The polymer of claim 10, wherein R¹ and R² ineach occurrence is independently H or an organic radical selected fromthe group consisting of lower alkyl and lower cycloalkyl.
 12. Thepolymer of claim 10, wherein R¹ and R² are both H, and Y is selectedfrom the group consisting of 3, 4, 5, 6, 7, 8, 9, and
 10. 13. Thepolymer of claim 10 having the structure:

wherein at least one of R¹ or R² on C_(α) is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, andy is at least one.
 14. The polymer of claim 13, wherein each of R¹ andR² on C_(α) is independently selected from the group consisting ofalkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl.
 15. The polymerof claim 13, wherein all other non-C_(α) R¹ and R² variables are H. 16.The polymer of claim 13 wherein at least one of R¹ or R² on C_(α) islower alkyl or lower cycloalkyl.
 17. The polymer of claim 13, wherein R²on C_(α) is H.
 18. The polymer of claim 17, wherein R¹ on C_(α) isselected from the group consisting of methyl, ethyl, propyl, isopropyl,butyl, isobutyl, pentyl, cyclopentyl, hexyl, and cyclohexyl.
 19. Thepolymer of claim 13 having the structure:

wherein R¹ and R² is each independently alkyl or cycloalkyl.
 20. Thepolymer of claim 13, having the structure:

wherein R¹ is alkyl or cycloalkyl and and R² is H.
 21. The polymer ofclaim 19 wherein R¹ and R² are each independently either methyl orethyl.
 22. The polymer of claim 19, wherein R¹ and R² are the same. 23.The polymer of claim 1, wherein said polymer possesses a hydrolyticallystabilized maleimide ring.
 24. The polymer of claim 8 having thestructure:

wherein R¹ and R² is each independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, butare not both H, and y is at least
 2. 25. The polymer of claim 24,wherein R¹ and R² is each independently H, lower alkyl or lowercycloalkyl.
 26. The polymer of claim 25, wherein R¹ and R² is eachindependently selected from the group consisting of H, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl, andcyclohexyl.
 27. The polymer of claim 24, wherein R² is H.
 28. Thepolymer of claim 10, having the structure:

wherein at least one of R¹ and R² attached to C_(γ) is selected from thegroup consisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl. 29.The polymer of claim 28, wherein at least one of R¹ and R² attached toC_(γ) is alkyl or cycloalkyl and all other R¹ and R² variables are H.30. The polymer of claim 28, wherein one of the R¹ variables attached toC_(α) or C_(β) is alkyl or cycloalkyl, and all other R¹ and R² variablesare H.
 31. The polymer of claim 2, wherein X is a saturated cyclic oralicyclic hydrocarbon chain.
 32. The polymer of claim 31, wherein X hasthe structure:

and CYC_(a) is a cycloalkylene group having “a” ring carbons, where thevalue of “a” ranges from 3 to 12; p and q are each independently 0 to20, and p+q+a≦20, R¹, in each occurrence, is independently H or anorganic radical that is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 33. The polymer of claim 32, wherein p and q areeach independently selected from the group consisting of 0, 1, 2, 3, 4,5, 6, 7, and
 8. 34. The polymer of claim 32, wherein R¹, in eachoccurrence, is independently H or an organic radical that is eitherlower alkyl or lower cycloalkyl, and R², in each occurrence, isindependently H or an organic radical that is either lower alkyl orlower cycloalkyl.
 35. The polymer of claim 32, wherein a is selectedfrom the group consisting of 5, 6, 7, 8 and
 9. 36. The polymer of claim35, wherein a is 6 and CYC_(a) is a 1,1-, 1,2-, 1,3- or 1,4-substitutedcyclohexyl ring.
 37. The polymer of claim 32, wherein p and q eachindependently range from 0 to
 4. 38. The polymer of claim 36, whereinthe substituents on said substituted cyclohexyl ring are cis.
 39. Thepolymer of claim 36, wherein the substituents on said substitutedcyclohexyl ring are trans.
 40. The polymer of claim 32, wherein R¹ andR² are H in every occurrence.
 41. The polymer of claim 34, having thestructure:

wherein q and p each independently range from 0 to
 6. 42. The polymer ofclaim 41, wherein q ranges from 0 to 6 and p is zero.
 43. The polymer ofclaim 34, having the structure:

wherein q and p each independently range from 0 to 6, and thesubstituents on the cyclohexylene ring are either cis or trans.
 44. Thepolymer of claim 32, wherein CYC_(a) is bicyclic or tricyclic.
 45. Thepolymer of claim 44, wherein CYC_(a) is selected from the groupconsisting of:

and said ring substituents are positioned at any available position onthe bi or tricyclic ring.
 46. The polymer of claim 1 having thestructure:

wherein X and b are as previously defined, b′ is 0 or 1, and X′ is ahydrolytically stable linker comprising at least 3 contiguous saturatedcarbon atoms.
 47. The polymer of claim 1, wherein POLY is selected fromthe group consisting of a poly(alkylene oxide), poly(vinyl pyrrolidone),poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), andpoly(oxyethylated polyol).
 48. The polymer of claim 47, wherein POLY isa poly(alkylene oxide).
 49. The polymer of claim 48, wherein POLY is apoly(ethylene glycol).
 50. The polymer of claim 49, wherein thepoly(ethylene glycol) is terminally capped with an end-capping moiety.51. The polymer of claim 50, wherein the end-capping moiety isindependently selected from the group consisting alkoxy, substitutedalkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substitutedalkynyloxy, aryloxy, and substituted aryloxy.
 52. The polymer of claim51, wherein the end-capping moiety is selected from the group consistingof methoxy, ethoxy, and benzyloxy.
 53. The polymer of claim 49, whereinthe poly(ethylene glycol) has a nominal average molecular mass of fromabout 100 daltons to about 100,000 daltons.
 54. The polymer of claim 53,wherein the poly(ethylene glycol) has a nominal average molecular massof from about 1,000 daltons to about 50,000 daltons.
 55. The polymer ofclaim 54, wherein the poly(ethylene glycol) has a nominal averagemolecular mass of from about 2,000 daltons to about 30,000 daltons. 56.The polymer of claim 46, wherein said POLY is linear and the polymer ishomobifunctional.
 57. The polymer of claim 49, wherein saidpoly(ethylene glycol) has a structure selected from the group consistingof linear, branched and forked.
 58. The polymer of claim 26, whereinsaid poly(ethylene glycol) comprises the structure:Z-(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about 10 to about 4000, and Zcomprises a moiety selected from the group consisting of hydroxy, amino,ester, carbonate, aldehyde, aldehyde hydrate, acetal, ketone, ketonehydrate, ketal, alkenyl, acrylate, methacrylate, acrylamide, sulfone,thiol, carboxylic acid, isocyanate, isothiocyanate, hydrazide, urea,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,alkoxy, benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein.59. The polymer of claim 1, corresponding to the structure:

wherein: d is an integer from 3 to about 100, and R is a residue of acentral core molecule having 3 or more hydroxyl groups, amino groups, orcombinations thereof.
 60. The polymer of claim 59, wherein d is aninteger from 3 to about
 12. 61. The polymer of claim 1, wherein POLY isa multi-arm polymer segment, and said polymer corresponds to thestructure:

where PEG is —(CH₂CH₂O)_(n)CH₂CH₂—, M is:

and m is selected from the group consisting of 3, 4, 5, 6, 7, and
 8. 62.A water-soluble polymer having the structure:

wherein POLY is a water-soluble polymer segment, b is 0 or 1, X is ahydrolytically stable linker comprising at least 3 contiguous saturatedcarbon atoms, and said polymer is absent aromatic groups and esterlinkages.
 63. The polymer of claim 62, wherein X is a saturated acyclic,cyclic or alicyclic hydrocarbon chain having a total of about 3 to about20 carbon atoms.
 64. The polymer of claim 62, wherein X is a saturatedacyclic, cyclic, or alicyclic hydrocarbon chain having a total number ofcarbon atoms selected from the group consisting of 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and
 20. 65. The polymer of claim64, wherein X is a saturated acyclic, cyclic, or alicyclic hydrocarbonchain having a total number of carbon atoms selected from the groupconsisting of: from about 3 to about 20, from about 4 to about 12, fromabout 4 to about 10, and from about 5 to about 8 atoms.
 66. The polymerof claim 63, wherein X is a linear saturated acyclic hydrocarbon chain.67. The polymer of claim 63, wherein X is a branched saturated acyclichydrocarbon chain.
 68. The polymer of claim 67, wherein X is branched atthe carbon α to the maleimidyl group.
 69. The polymer of claim 67,wherein X is branched at the carbon β to the maleimidyl group.
 70. Thepolymer of claim 67, wherein X is branched at the carbon γ to themaleimidyl group.
 71. The polymer of claim 63, having the structure:

wherein: y is an integer from 1 to about 20; R¹, in each occurrence, isindependently H or an organic radical that is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl, andR², in each occurrence, is independently H or an organic radical that isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 72. The polymer of claim 62, wherein X is asaturated cyclic or alicyclic hydrocarbon moity.
 73. The polymer ofclaim 72, wherein X has the structure:

and CYC_(a) is a cycloalkylene group having “a” ring carbons, where thevalue of “a” ranges from 3 to 12; p and q are each independently 0 to20, and p+q+a≦20, R¹, in each occurrence, is independently H or anorganic radical that is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 74. The polymer of claim 73, wherein p and q areeach independently selected from the group consisting of 0, 1, 2, 3, 4,5, 6, 7, and
 8. 75. The polymer of claim 73, wherein R¹, in eachoccurrence, is independently H or an organic radical that is eitherlower alkyl or lower cycloalkyl, and R², in each occurrence, isindependently H or an organic radical that is either lower alkyl orlower cycloalkyl.
 76. The polymer of claim 73, wherein a is selectedfrom the group consisting of 5, 6, 7, 8 and
 9. 77. The polymer of claim76, wherein a is 6 and CYC_(a) is a 1,1-, 1,2-, 1,3- or 1,4-substitutedcyclohexylene ring.
 78. The polymer of claim 77, having the structure:

wherein q and p each independently range from 0 to 6, and thesubstituents on said cyclohexylene ring are either cis or trans.
 79. Thepolymer of claim 77, having the structure:

wherein q and p each independently range from 0 to 6, and thesubstituents on said cyclohexylene ring are either cis or trans.
 80. Thepolymer of claim 73, wherein CYC_(a) is bicyclic or tricyclic.
 81. Amethod for forming a maleimide terminated polymer, said methodcomprising the steps of: a. reacting POLY-[O]_(b)—C(O)-LG (IX) withNH₂—X—NH₂ (XII) under conditions effective to formPOLY-[O]_(b—)C(O)—H₂N—X—NH₂ (X), and b. convertingPOLY-[O]_(b)—C(O)—H₂N—X—NH₂ (X) into POLY-[O]_(b)—C(O)—HN—X-MAL (II),wherein: POLY is a water-soluble polymer segment, b is 0 or 1, X is ahydrolytically stable linker comprising at least 3 contiguous saturatedcarbon atoms, LG is a leaving group, MAL is maleimide, and saidmaleimide terminated polymer is absent aromatic groups and esterlinkages.
 82. The method of claim 81, wherein said LG is selected fromthe group consisting of halide, N-hydroxysuccinimide,N-hydroxybenzotriazole, para-nitrophenolate.
 83. The method of claim 81,wherein one of said amino groups in said NH₂—X—NH₂ reagent is inprotected form.
 84. The method of claim 81, wherein said reacting stepis carried out in an organic solvent selected from the group consistingof acetonitrile, chloroform, dichloromethane, benzene, toluene, xylene,acetone, tetrahydrofuran (THF), dimethylformamide (DMF), anddimethylsulfoxide.
 85. The method of claim 81, wherein said reactingstep is conducted under an inert atmosphere.
 86. The method of claim 81,wherein said reacting step is conducted at a temperature in the range ofabout 0 to 100° C.
 87. The method of claim 81, wherein said reactingstep is conducted in the presence of a base selected from the groupconsisting of triethylamine, pyridine, 4-(dimethylamino)pyridine, andsodium carbonate.
 88. The method of claim 83, further comprising aftersaid reacting step, deprotecting the amino group inPOLY-[O]_(b)—C(O)—H₂N—X—NH₂.
 89. The method of claim 81, furthercomprising the step of purifying the product from step (a) prior to saidconverting step.
 90. The method of claim 89, wherein said purifyingcomprises purifying the product by column chromatography.
 91. The methodof claim 89, wherein said purifying comprises purifying the product byion exchange chromatography.
 92. The method of claim 81, wherein saidconverting step comprises reacting POLY-[O]_(b)—C(O)—H₂N—X—NH₂ with areagent selected from the group consisting ofN-methoxycarbonylmaleimide,exo-7-oxa[2.2.1]bicycloheptane-2,3-dicarboxylic anhydride, and maleicanhydride under conditions suitable for formingPOLY-[O]_(b)—C(O)—H₂N—X-MAL in a reaction mixture.
 93. The method ofclaim 92, wherein when said reagent is N-methoxycarbonylmaleimide, andsaid converting step is carried out in water or an aqueous mixture ofwater and a water miscible solvent.
 94. The method of claim 92, whereinsaid reagent is maleic anhydride, and said converting step comprisesreacting POLY-[O]_(b)—C(O)—H₂N—X—NH₂ with maleic anhydride underconditions effective to form POLY-[O]_(b)—C(O)—NH—X—NH—C(O)CH═CHCOOH(XI) as an intermediate, and said method further comprises: heating saidPOLY-[O]_(b)—C(O)—H₂N—X—NH—C(O)CH═CHCOOH under conditions effective topromote cyclization by elimination of water to formPOLY-[O]_(b)—C(O)—NH—X-MAL.
 95. The method of claim 92, furthercomprising recovering said POLY-[O]_(b)—C(O)—H₂N—X-MAL from the reactionmixture.
 96. The method of claim 95, wherein said recoveredPOLY-[O]_(b)—C(O)—H₂N—X-MAL has a purity of greater than about 80%. 97.The method of claim 81, wherein X is a saturated acyclic, cyclic oralicyclic hydrocarbon chain having a total of about 3 to about 20 carbonatoms.
 98. The method of claim 81, wherein X is a linear saturatedacyclic hydrocarbon chain.
 99. The method of claim 81, wherein X is abranched saturated acyclic hydrocarbon chain.
 100. The method of claim99, wherein X is branched at the carbon α to the maleimidyl group. 101.The method of claim 99, wherein X is branched at the carbon β to themaleimidyl group.
 102. The method of claim 99, wherein X is branched atthe carbon γ to the maleimidyl group.
 103. The method of claim 81,wherein said NH₂—X—NH₂ corresponds to the structure:

wherein y is an integer from 1 to about 20; R¹, in each occurrence, isindependently H or an organic radical that is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkylenecycloalkyl, and substituted alkylenecycloalkyl andR², in each occurrence, is independently H or an organic radical that isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 104. The method of claim 81, wherein said NH₂—X—NH₂corresponds to the structure:

and CYC_(a) is a cycloalkylene group having “a” ring carbons, where thevalue of “a” ranges from 3 to 12; p and q are each independently 0 to20, and p+q+a≦20, R¹, in each occurrence, is independently H or anorganic radical that is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 105. A conjugate formed by reaction of abiologically active agent with the polymer of claim
 1. 106. A conjugatecomprising the following structure:

wherein: POLY is a water-soluble polymer segment, b is 0 or 1, X is ahydrolytically stable linker comprising at least 3 contiguous saturatedcarbon atoms, “POLY-[O]_(b)—C(O)—NH—X-” is absent aromatic groups andester linkages, and “—S-biologically active agent” represents abiologically active agent comprising a thiol (—SH) group.
 107. Acomposition comprising the conjugate of claim 106, wherein saidcomposition comprises a single polymer conjugate species.
 108. Aconjugate comprising the following structure:

wherein: POLY is a water-soluble polymer segment, b is 0 or 1, X is ahydrolytically stable linker comprising at least 3 contiguous saturatedcarbon atoms, “POLY-[O]_(b)—C(O)—NH—X-” is absent aromatic groups andester linkages, and “—NH-biologically active agent” represents abiologically active agent comprising an amino group.
 109. A method forforming a polymer conjugate, said method comprising contacting abiologically active agent comprising a reactive thiol group,“HS-biologically active agent”, with a polymer of claim 1, underconditions effective to promote formation of a polymer conjugate havingthe structure:


110. The method of claim 109, wherein said contacting step is carriedout at pHs ranging from about 6.0 to about 8.0.
 111. The method of claim110, wherein said polymer conjugate is formed in a reaction mixture, andsaid method further comprises after said contacting step, isolating saidpolymer conjugate from said reaction mixture.
 112. A hydrogel formedusing the water-soluble polymer of claim
 1. 113. A hydrogel formed usingthe water soluble polymer of claim
 59. 114. A hydrogel formed using thewater soluble polymer of claim
 61. 115. A water soluble polymer havingthe structure:

wherein: POLY is a water-soluble polymer segment, X is a hydrolyticallystable linker that is a saturated cyclic or alicyclic hydrocarbon chainhaving a total of about 3 to about 20 carbon atoms, and said polymer isabsent aromatic groups and ester linkages.
 116. The polymer of claim115, wherein X has the structure:

and CYC_(a) is a cycloalkylene group having “a” ring carbons, where thevalue of “a” ranges from 3 to 12; p and q are each independently 0 to20, and p+q+a≦20, R¹, in each occurrence, is independently H or anorganic radical that is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl,alkylenecycloalkyl, and substituted alkylenecycloalkyl, and R², in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkylenecycloalkyl, and substitutedalkylenecycloalkyl.
 117. The polymer of claim 116, wherein p and q areeach independently selected from the group consisting of 0, 1, 2, 3, 4,5, 6, 7, and
 8. 118. The polymer of claim 116, wherein R¹, in eachoccurrence, is independently H or an organic radical that is selectedfrom the group consisting of lower alkyl, lower cycloalkyl, and loweralkylenecycloalkyl, and R², in each occurrence, is independently H or anorganic radical that is selected from the group consisting of loweralkyl, lower cycloalkyl, and lower alkylenecycloalkyl.
 119. The polymerof claim 116, wherein a is selected from the group consisting of 5, 6,7, 8 and
 9. 120. The polymer of claim 116, wherein the substituents onCYC_(a) are cis.
 121. The polymer of claim 116, wherein the substituentson CYC_(a) are trans.
 122. The polymer of claim 119, wherein a is 6 andCYC_(a) is a 1,1-, 1,2-, 1,3-or 1,4-substituted cyclohexyl ring. 123.The polymer of claim 116, wherein p and q each independently range from0 to
 4. 124. The polymer of claim 116, wherein R¹ and R² are H in everyoccurrence.
 125. The polymer of claim 116, having the structure:

wherein q and p each independently range from 0 to
 6. 126. The polymerof claim 116, wherein q ranges from 0 to 6 and p is zero.
 127. Thepolymer of claim 116, having the structure:

wherein q and p each independently range from 0 to
 6. 128. The polymerof claim 116, wherein CYC_(a) is bicyclic or tricyclic.
 129. The polymerof claim 116, wherein CYC_(a) is selected from the group consisting of:

and said ring substituents are positioned at any available position onthe bi or tricyclic ring.
 130. A method for forming a maleimideterminated polymer, said method comprising: a. reactingPOLY-[O]_(b)—C(O)-LG with H₂N—X-MAL under conditions effective to formPOLY-[O]_(b)—C(O)—HN—X-MAL, wherein: POLY is a water-soluble polymersegment, b is 0 or 1, X is a hydrolytically stable linker comprising atleast 3 contiguous saturated carbon atoms, LG is a leaving group, MAL ismaleimide, and said maleimide terminated polymer is absent aromaticgroups and ester linkages.